Abnormal regulation of specific genes in highly differentiated tissues constitutes the underlying defect in the thalassemia syndromes as well as in other genetic disorders. The overall aim of this project is to elucidate the molecular interactions between tissue specific nuclear proteins and the DNA sequences encompassing the globin genes which result in precise regulation of globin gene expression during erythroid differentiation and developmental hemoglobin switching. Several unique features of the avian erythroid system, including a well-characterized animal model of embryonic globin gene transcriptional activation in adult erythroid tissues, make this an ideal system in which to study these interactions in a physiologic setting. Putative tissue-specific and developmentally stage-specific nuclear factors which regulate embryonic globin gene expression will be identified in normal embryonic and adult erythroid tissues as well as in cultured erythroid cells by a series of in vitro DNA- protein binding assays and functional assays. These protein factors will be purified by standard biochemical approaches, their functional characteristics will be assayed in in vitro transcription systems, and attempts will be made to clone the gene(s) coding for such nuclear regulatory factors. Cis-acting DNA sequences and modifications which affect binding of the putative trans-acting factors will be studied in DNA mediated gene transfer assays in which globin fusion gene constructs will be introduced into primary and cultured avian erythroid cells. The regulatory role of various sequences within and around the globin genes as well as the regulatory role of site specific DNA methylation will be assayed. In addition, in vitro assays will be employed to identify alterations in nucleosomal subunits that are characteristic of active chromatin. The long term goal of this project is to improve the understanding of the molecular mechanisms which control cellular differentiation at the level of specific gene expression. Such knowledge should be directly applicable to hereditary diseases such as thalassemia and sickle cell anemia in which the ability to alter expression of specific globin genes could overcome the pathophysiology of these disorders. Additionally, an understanding of gene regulatory processes should offer hope for designing more effective therapies for other hereditary diseases as well.
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