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 erythroiddifferentiation at the proerythroblast level. We recently demonstrated that the gene that is defective in thezebrafish moonshine mutant is the transcriptional intermediary factor 1 gamma (TIF1gamma). The TIF1 family hasmultiple domains including a PhD finger, ring finger and bromo domain, and are thought to bridge DNAbinding proteins to other chromatin factors. One published role for TIFs is to modulate transcriptionregulation by nuclear receptors. TIF1gamma is localized to novel nuclear bodies, and to date no signaling factorshave been found to interact with TIF1gamma during erythropoiesis. Recently TIF1gamma has been shown to interactwith SMAD factors and regulate epithelial fate during early embryogenesis in frogs, establishing a hypothesisthat TGFbeta signaling is abnormal is moonshine mutants. Here we plan to do an extensive characterization ofgene expression in moonshine mutants. We plan to sort erythroid progenitors at the onset of the moonshinephenotype, and compare gene expression profiles to wildtype cells, and to other hemtopoietic mutants. Theeffect of overexpression of TIF1gamma will be evaluated in zebrafish embryos. We plan to use chromatinimmunoprecipitation as a method to find targets of TIF1gamma, and will purify TIF1gamma associated proteins using anin vivo biotinylation and streptavidin purification strategy. Particular attention will be given to SMAD factorsthat might interact with TIF1gamma in erythroid cells at a genetic level. Targeted lesion detection will be used inzebrafish to find an allelic series of moonshine mutants, with mutations in every functional domain ofmoonshine. Finally, we will undertake a suppressor screen for zebrafish mutants that modify the moonshinephenotype. This should lead to a better understanding of the pathways that moonshine controls, and willhave an impact on diseases such as sickle cell anemia and thalassemia.
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