| Abstract |
GATA1 is hematopoietic transcription factor that is known to be critical for the differentiation of several hematopoietic lineages, including erythroid cells, megakaryocytes, mast cells, eosinophils and dendritic cells. GATA1 exists as a full length protein and as a short (GATA1s) isoform that lacks the N-domain. Importantly, mutations resulting in the exclusive expression of GATA1s are implicated in human hematopoietic disorders like Diamond Blackfan Anemia (DBA) and acute megakaryoblastic leukemia (AMKL) associated with Trisomy 21 in Down Syndrome. The goal of the study is to investigate the utility of two mouse models bearing a biotin tag knocked-in the GATA1 gene locus to study the functions of GATA1 and GATA1s during the formation of the aforementioned lineages. Specifically, we tested whether the two knock-in mouse models expressed full length biotin-tagged GATA1 or biotin-tagged GATA1s in these lineages, when crossed with transgenic mice ubiquitously expressing the biotin ligase BirA which is required for biotinylation of the short biotinylatable tag fused to GATA1. In assessing biotinylation tagging of these two GATA1 isoforms in hematopoietic lineages, I optimized the purification of dendritic cells and mast cells from the bone marrow of adult mice, whereas eosinophils were isolated from the spleen of IL-5 transgenic mice. In contrast, megakaryocytes were isolated from E12.5 fetal livers. My study suggests that megakaryocytes and dendritic cells express GATA1 at such levels that it can be detected in crude protein extracts. Detection of BirA expression also suggests that bioGATA1 and bioGATA1s are likely to be in vivo biotinylated in these lineages. Eosinophils were also found to express detectable levels of tagged GATA1, however it was not possible to detect BirA expression or biotinylation. Mast cells, as previously described, do not appear to express GATA1 unless they are stimulated with IL-3. Thus, in this study I have successfully applied methods for the isolation of the hematopoietic cells of interest from mouse bone marrow or fetal liver cells and have identified megakaryocytes, dendritic cells and possibly eosinophils as lineages where the biotin tag knock-in GATA1 and GATA1s mouse models would be useful in studying their function.
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Characterization of GATA1 transcription factor functions in murine hematopoiesis
