Hemoglobin production is an evolutionarily conserved process that is specific to the red blood cell lineage. We have utilized the zebrafish as a model for studying this process. Our analysis of mutant fish with a hypochromic microcytic anemia has provided key insights into factors that regulate hemoglobin synthesis. These factors fall into four categories: the production of heme, iron utilization, globin expression, and iron sulfur cluster production. We have found several novel genes involved in hemoglobin production, including ferroportin, mitoferrin, and glutaredoxin 5. The first two of these factors were found to be mutated in patients with human disorders, establishing the zebrafish system as a model for human disease. To date, we have uncovered over eight complementation groups of hypochromic anemia. In this proposal, we plan to extend our observations by characterizing the globin locus of the zebrafish, including its associated chromatin structure. Regulatory elements will also be examined. The zebrafish locus control region (LCR) is representative of an ?LCR. In transgenics, bringing the zebrafish LCR into apposition with globin genes drives high level of expression within erythroid cells. Transgenic fish are being created with human globin BACs to evaluate conservation of regulatory elements. The mutant zinfandel lacks embryonic globin production, but recovers and has normal adult hemoglobin formation. This gene was mapped to the globin locus on chromosome 3 and no globin coding regions are mutated. A high-resolution positional cloning project is underway to define this novel embryonic-specific globin regulatory mutation. We also developed a method for genetically analyzing chromatin factors that regulate hemoglobin production. The technology of morpholinos in which antisense oligonucleotides against specific genes are injected into the one cell embryo can knock-down gene function and will reveal changes in erythropoiesis. A compendium of over 300 chromatin remodeling factors that are orthologs of human genes has been created, and each will be studied by gene knock-down in zebrafish embryos. The effect of gene knock-down on erythroid gene expression (including LCR activation and globin gene expression) will be evaluated. These factors will be studied genetically in the zebrafish and interactions with specific transcription factors will be investigated by double knock-down and overexpression studies. Using human erythroid cells, we plan to biochemically purify a complex of chromatin factor orthologs found through this genetic screen. The understanding of chromatin factors that affect hemoglobin production will have a tremendous impact on our understanding of hemoglobin production and for the treatment of patients with thalassemia, sickle cell anemia, and globin mutations.
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