An understanding of the factors that control tissue-specific gene regulation is a prerequisite to attempts to understand and treat at the molecular level the defects in a variety of common human genetic disorders, including the thalassemia syndromes. The overall aim of this project is to elucidate molecular interactions between the cis-acting DNA sequences and cognate trans-acting nuclear regulation factors that control globin gene expression during erythroid differentiation and developmental globin gene switching. Several unique features of the of the avian erythroid system, including a well characterized animal model of reversed hemoglobin switching by transcriptional activation of embryonic globin genes in adult erythroid cells, coupled with mounting evidence for evolutionary conservation of globin gene control elements, make this a desirable system in which to study these regulatory processes in a physiologically relevant mariner. To achieve the aims of this project, cis-acting DNA sequences and modifications that have been shown to be involved in embryonic globin gene regulation will be fully characterized by in vitro mutagenesis and functional assays through DNA-mediated gene transfer into primary or cultured erythroid cells and transgenic animals. The role of chromosomal integration, chromatin structural features and DNA methylation in the activity of putative developmental silencer elements will be determined by analysis of DNA modifications, chromatin structure and DNA-protein interactions in intact cultured cells and primary erythroid cells in vivo and in biochemically defined in vitro assays. Putative stage-specific nuclear factors that regulate developmental expression of embryonic globin gene expression will be isolated from cultured and primary erythroid cells and characterized by in vitro DNA binding assays corroborated by in vivo DNA-protein binding assays. These protein factors will then be purified by standard biochemical methods to allow functional analysis in in vitro transcription assay systems. At the same time, attempts will be made to clone the genes for those factors that appear to be functionally relevant in order to allow production of large quantities for mechanistic studies as well as to allow testing of their precise function in vivo by surrogate genetic assays. The ultimate goal of this project is to define at a molecular level the mechanism(s) by which embryonic globin genes are silenced in adult erythroid cells and the mechanism by which pharmacologic agents can reverse this developmental silencing in vivo. The knowledge gained from this project should be directly applicable to therapeutic attempts to overcome the pathophysiologic defects in the majority of beta-type thalassemia syndromes and in sickle cell anemia. Beyond this direct application, the knowledge gained about differential tissue-specific gene regulation could facilitate the development of rational therapy for a host of other common diseases that result from genetic defects.
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