A detailed understanding of the mechanisms that control globin gene expression is a prerequisite to the development of safe and effective molecular therapy of common genetic blood disorders including sickle cell anemia and beta-thalassemia. The overall objective of this proposed project is to elucidate genetic and epigenetic mechanisms that regulate transcription of globin genes in erythroid cells. In order to achieve this objective, the following specific aims are prepared: (1) to determine the mechanism by which DNA methylation suppresses transcription of cytosine-rich globin genes; (2) to determine the role of specific histone and non-histone protein acetylation in the developmental regulation of beta-type globin gene transcription; (3) to elucidate the mechanisms by which short-chain fatty acids stimulate transcription of embryonic/fetal globin genes in adult erythroid cells; and (4) to determine the role of 3' flanking sequences in the developmental regulation of the human epsilon globin gene in a transgenic mouse model. The availability of large quantities of pure stage-specific erythroid cells in the avian erythroid model system will facilitate biochemical characterization of histone and non-histone protein acetylation and isolation of nuclear factors involved in DNA methylation mediated repression of transcription. The availability of a primary erythroid cell gene transfection assay will allow functional testing of factors that are shown to correlate with transcriptional activation or repression in the same stage-specific erythroid cells in vivo. A transgenic mouse model of human epsilon (epsilon) globin gene expression will facilitate studies of the role of 3' flanking sequences in the developmental silencing of transcription and will allow extension of the studies in the avian embryonic rho-globin gene to the regulation of its human counterparts. It is expected that the experimental plan prepared in this application will increase the understanding of the role and mechanisms of DNA methylation and histone acetylation and demonstrate how these epigenetic processes interact in the control of developmental globin gene switching. This knowledge could ultimately facilitate attempts to develop new treatments for sickle cell anemia and beta-thalassemia. Because of the well-recognized general importance of these epigenetic regulatory processes in controlling expression of many higher eukaryotic genes, including tumor expression genes, it is anticipated that the new knowledge gained from this project will provide valuable new insights into the fundamental regulatory mechanisms of many genes that are critical in other human diseases as well.
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