It is estimated that more than 3% of the world's population carry mutations in the 2-globin gene locus that cause more or less severe cases of anemia. One of the best known and characterized of these mutations is an amino acid change in the adult 2-globin chain that causes sickle cell disease (SCD). Other mutations lead to thalassemias, which are characterized by reduction in globin gene synthesis. Currently, there is no satisfactory treatment for hemoglobinopathies that will benefit all patients. The 2-globin gene locus consists of five genes that are competitively regulated by a locus control region (LCR) during development. The LCR is composed of five DNase I hypersensitive (HS) sites that are spread over more than 10 kbp of DNA and which act together to mediate high-level globin gene expression. Despite advances in understanding globin gene regulation over the last three decades, how the LCR operates to mediate high level globin gene expression is not understood. A detailed knowledge of the molecular mechanisms leading to stage specific expression of the globin genes will aid the development of novel and more broadly applicable therapies for hemoglobinopathies. This is best illustrated by the fact that expression of the fetal 3-globin in adults homozygous for the sickle cell mutation ameliorates the severe form of the disease. The development of strategies that silence the mutant 2-globin gene and at the same time activate expression of the 3-globin genes would offer novel therapies for a variety of hemoglobinopathies. Clearly, a detailed understanding of how the genes are competitively regulated by the LCR is crucial for the development of therapies that target or take advantage of LCR mediated mechanisms of globin gene regulation. Approach: The goal of this application is to decipher the mechanism(s) by which the LCR regulates 2-globin gene expression. It is proposed that the LCR serves as the primary attachment and assembly site for elongation active transcription complexes in the 2-globin gene locus. The elongation active transcription complexes are transferred to globin gene promoters in a developmental stage specific manner. This hypothesis will be addressed in transgenic mice by examining if the LCR is sufficient to form transcription foci in erythroid cells and how transcription factor upstream stimulatory factor (USF) in conjunction with erythroid specific transcription factors regulates this process (Specific Aim 1). Furthermore, to analyze the mechanism of transcription complex recruitment and assembly, components of transcription complexes recruited to the LCR and to the adult 2-globin gene promoter will be characterized using a novel in vitro assembly assay as well as using chromatin immunoprecipitation (ChIP) in intact cells (Specific Aim 2). We will map accessibility in the LCR and the rest of the 2-globin gene locus during differentiation using a novel approach, called MAPit, designed to assay protein DNA interactions at high resolution on single molecules (Specific Aim 3). Finally, to identify cis-regulatory DNA elements that mediate interactions between the LCR and the globin genes we will express zinc finger DNA binding domains that neutralize transcription factor binding sites and examine the effect of these proteins on globin gene expression and LCR globin gene interactions (Specific Aim 4).
The goal of this study is to increase our knowledge of how the 2-globin genes are regulated by the locus control region and to develop new tools that could aid in finding better cures for hemoglobinopathies. The major focus of this application is to determine the role of transcription factors and cis-regulatory elements in the high-level expression of the 2-globin gene. Interfering with activities that mediate high-level expression of the adult 2-globin gene could lead to increased expression of the therapeutic 3-globin genes in patients with sickle cell disease.
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