Prior to becoming a full time faculty member at the University of South Alabama, I completed four years of post-doctoral training, performing research in the field of hemoglobin switching. The goal for my research was the identification of new agents with fetal hemoglobin inducibility for the treatment of sickle cell patients. One such agent, butyric acid induces fetal hemoglobin both in vivo and in vitro. Recent data suggest butyrate may require specific DNA sequences, in the promoter region of several genes, in order to induce gene expression. The role of nuclear trans-acting factors in this process has not been defined. With regards to the effects of this agent in the gamma globin promoter, my studies in transgenic mice demonstrated the inability of butyrate to reactive a totally silenced gamma gene. Utilizing detailed truncation analysis of the gamma globin promoter in transgenic animals, new insights were gained into the cis sequences involved in the induction of gamma gene expression by butyric acid. Subsequent studies in vitro in mouse erythroleukemia cells suggested the sequence between nucleotides-822 and -893 is necessary for modulation of gamma gene activity, following induction with butyrate. In addition, gel shift analysis indicates sequence specific proteins bind in this region. My hypothesis is that butyrate induces gamma gene expression through modifications of sequence specific nuclear proteins which bind in the upstream promoter region. This hypothesis will be tested by completing the following Specific Aims: 1) To establish a transient assay system to test the ability of butyric acid to induce gamma gene expression in the presence of the beta-globin gene. 2) To study the ability of the identified butyrate response element to confer butyrate inducibility to a heterologous promoter. 3) Isolation of the cDNA(s) encoding the butyrate response element DNA-binding protein(s) using an in vivo one hybrid system. 4) Establish a prokaryotic expression system for protein synthesis and purification. 5) Define the characteristics of the sequence specific protein by structure-function analysis. The information gained by this analysis will impart new insights into the mechanism by which butyrate induces gamma gene expression. My institution has provided an environment conducive to strong basic research activities and sufficient opportunity for meaningful collaborative efforts.
- National Heart, Lung, and Blood Institute: $133,650.00
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