A human erythroid specific transcriptional enhancer lOkb upstream of the epsilon globin gene has been identified. DNA sequences including this enhancer have been shown to confer high level position independent transcription of the human Beta globin gene in transgenic mice and are deleted in the human gamma-delta-beta thalassemias. The location of this enhancer element suggests it maintains openness of the entire Beta-like gene domain to transcriptional factors. Thus this sequence and the proteins which activate it are required for Beta-like globin gene transcription. The goal of our studies is to identify and purify erythroid specific enhancer activating proteins and identify the minimal sequence required for position independent erythroid specific transcriptional enhancement. To this end the following studies will be undertaken: l-Investigation of in vivo and in vitro DMS interference and DNase protection studies to identify ubiquitous and erythroid specific DNA sequence motifs and cognate enhancer binding proteins. 2-Electrophoretic gel mobility shift assays will be used in conjunction with chromatographic fractionation of erythroid and non-erythroid nuclear extracts to identify and purify enhancer binding proteins. 3-Stable cell lines containing the enhancer with/without additional epsilon upstream DNA driving a reporter gene will be produced in order to identify the minimal enhancer sequence required for position independent erythroid specific high level gene transcription. 4-Subcloning of restriction fragments and Bal 31 deletions will be made to identify the minimal sequence required for erythroid specific expression. Identification of erythroid specific proteins binding the enhancer and maintaining its open chromatin conformation is required for understanding of transacting proteins and their role in alteration of chromatin structure and commitment to erythroid differentiation. Identification of the minimal sequence required for position independent erythroid specific transcriptional enhancement is necessary for construction of effective vectors for the genetic therapy of Beta thalassemia and sickle cell anemia.
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