Erythropoiesis is driven by an intrinsic transcriptional program that is modified by chromatin factors. Homozygous mutant moonshine mutants are severely anemic and have a complete block in erythroid differentiation at the proerythroblast level. We recently demonstrated that the gene that is defective in the zebrafish moonshine mutant is the transcriptional intermediary factor 1 gamma (TIF1gamma). The TIF1 family has multiple domains including a PhD finger, ring finger and bromo domain, and are thought to bridge DNA binding proteins to other chromatin factors. One published role for TIFs is to modulate transcription regulation by nuclear receptors. TIF1gamma is localized to novel nuclear bodies, and to date no signaling factors have been found to interact with TIF1gamma during erythropoiesis. Recently TIF1gamma has been shown to interact with SMAD factors and regulate epithelial fate during early embryogenesis in frogs, establishing a hypothesis that TGFbeta signaling is abnormal is moonshine mutants. Here we plan to do an extensive characterization of gene expression in moonshine mutants. We plan to sort erythroid progenitors at the onset of the moonshine phenotype, and compare gene expression profiles to wildtype cells, and to other hemtopoietic mutants. The effect of overexpression of TIF1gamma will be evaluated in zebrafish embryos. We plan to use chromatin immunoprecipitation as a method to find targets of TIF1gamma, and will purify TIF1gamma associated proteins using an in vivo biotinylation and streptavidin purification strategy. Particular attention will be given to SMAD factors that might interact with TIF1gamma in erythroid cells at a genetic level. Targeted lesion detection will be used in zebrafish to find an allelic series of moonshine mutants, with mutations in every functional domain of moonshine. Finally, we will undertake a suppressor screen for zebrafish mutants that modify the moonshine phenotype. This should lead to a better understanding of the pathways that moonshine controls, and will have an impact on diseases such as sickle cell anemia and thalassemia.
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