REGULATION OF THE HUMAN CD34 STEM CELL GENE

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CD34 is expressed specifically on human hematopoietic stem cells and not on mature blood cells. CD34+ cells can reconstitute normal hematopoiesis of all cell lines, and to date it represents the only well defined human stem cell marker; therefore, it is a model for stem cell gene expression. Previous studies have defined the promoter for CD34, as well as identified a large fragment 3' of the CD34 gene containing an enhancer which appears to direct specific expression of GD34 in cell lines. The goal of this project is to further define and analyze the control elements of CD34, and to characterize those elements of the CD34 promoter and enhancer which can direct stem cell expression of heterologous genes. The characterization of these elements could lead to the development of new vectors for gene therapy, as well as new understanding of the factors regulating genes in stem cells. Therefore, the specific aims are to characterize and study the regulatory elements of CD34 in the following steps: (l) To isolate and characterize the precise genomic sequences comprising the functional and tissue specific elements of the CD34 3' enhancer; (2) To demonstrate that the same elements which control CD34 gene expression in cell lines regulate its expression in normal hematopoietic cells, demonstrating that these elements regulate CD34 expression in transgenic mice and in normal human CD34+ cells; (3) To isolate the minimal CD34 promoter/enhancer which will direct expression of a reporter gene in stem cells; and (4) To characterize the transcription factor(s) which interact with the CD34 3' enhancer elements defined in aims (l) and (2) to control the expression of CD34. These studies should lead to a better understanding of the regulation of stem cell genes, as well as the identification of the transcription factors which regulate them. In addition, these experiments shall also define the minimal regulatory elements necessary to direct stem cell expression of foreign genes, which should lead to new applications in gene therapy.