The goals of this proposal are to define potentially distant DNA sequences involved in various aspects of regulation of the human globin genes; particularly, tissue and developmental stage- specificity and developmental switching. This represents a continuation of an already established research project in which potential control regions associated with the embryonic sigma- globin gene have been located as regions of erythroid-specific DNAseI hypersensitivity. The functional significance of these regions will be tested by introducing specific sigma-globin gene mutants into a range of cell lines representing various stages of erythroid development and assaying transcriptional activity by various types of RNA analysis, principally S1 and primer extension. To gain insight into the time scale within which particular control elements may operate during erythroid differentiation or development, the pattern of sigma-globin DNAseI hypersensitive sites characteristic of different functional states of the gene will be examined. This will make use of both erythroid cell lines and normal erythroid tissues obtained from different developmental stages. The combination of transfection experiments and analysis of DHS is also aimed at defining those control elements peculiar to sigma-globin, these implicated in activation of the entire beta- globin cluster. A novel experimental approach using a combination of cellular transfection and somatic cell hybridization is described, whose aim is to identify sigma-globin control elements required for the early stages of activation of the gene. One previously identified sigma-globin control element is associated with down-regulation of the gene following introduction into non-erythroid cell lines. This element may operate either in undifferentiated erythroid stem cells or may be involved in the developmental inactivation of the gene. It is proposed to test the hypothesis that the mechanism by which this element operates is based on transcriptional interference. The mechanisms by which this may be overcome in differentiated erythroid cells will be investigated. The interaction of defined globin control elements with regulatory molecules will be investigated both by in vivo competition assays and by in vitro """"""""footprinting"""""""" techniques.
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