The human ?-globin gene locus consists of five genes that are expressed in a developmental stage- and erythroid-specific manner and regulated by a locus control region (LCR) located far upstream of the genes. Mutations in the ?-globin gene locus are quite frequent in the human population and associated with mild to severe anemias. Most of these mutations are located within the coding region of the adult ?-globin gene or within regulatory sequences affecting expression of the adult gene. Among the most prominent of ?-globin associated diseases are sickle cell anemia (SCA) and ?- thalassemias. Current treatments for these diseases are unsatisfactory and much effort is being invested in developing novel forms of therapies. We anticipate that further knowledge of how the globin genes are activated during development will create new opportunities for the treatment of hemoglobinopathies. For example, recent evidence suggests that the regulatory elements of the globin locus, including the LCR and the gene promoters, come in close proximity during activation of gene expression. If it is understood how genes are brought into close proximity to the LCR it may be possible in the future to modulate these interactions in such a way that associations of the ?-globin genes are favored over that of the adult ?-globin gene in definitive erythroid cells. We propose to perform a comprehensive analysis of the composition and function of protein complexes containing transcription factors involved in globin gene regulation. In these studies we will utilize novel methodology, including expression of biotin tagged proteins in differentiating erythroid cells and in transgenic mice, to examine the role of transcription factors and associated co-factors in the regulation of chromatin accessibility, globin locus conformational changes, and recruitment of transcription complexes. Biotinylated transcription factors will be isolated from cells using streptavidin coated magnetic beads and subjected to mass-spectrometry for identifying associated proteins, to chromatin immunoprecipitation (ChIP) for identifying associated DNA sequences, and to a combined ChIP and chromosome conformation capture (3C) assay for identifying proteins that function in the context of a globin locus configuration in which the LCR and promoters are in close proximity.
The goal of this study is to provide a comprehensive understanding of the role of transcription factors involved in ?-globin gene regulation using novel methodology. Biotin-tagged transcription factors will be expressed in erythroid cells, isolated using streptavidin coated magnetic beads, and subjected to various biochemical and molecular assays to identify interacting proteins and DNA regions they bind to as well as to determine whether the proteins are part of and required for the formation of """"""""looped"""""""" chromatin configurations that bring regulatory elements of the ?-globin locus in close proximity.
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