| PU.1 is an Ets transcription factor that plays essential roles in hematopoietic cell fate decisions. It functions in a concentration-dependent manner to activate and repress target genes of specific lineages. PU.1 is required for myeloid and B cell commitment; however, it must be downregulated for erythroid or T cell development to occur. Inactivating mutations of PU.1 can lead to myeloid, B cell, or T cell leukemia in humans and mice. The central goal of this thesis project is to determine how the concentration of PU.1 affects the regulation of target genes in hematopoiesis. The central hypothesis is that a reduction in PU.1 concentration causes lineage-inappropriate gene expression in vitro and in vivo, leading to disproportionate differentiation of hematopoietic stem cells and a lineage identity crisis.;For this project, we utilized hypomorphic alleles of the gene encoding PU.1 (Sfpi1) generated by our lab and analyzed cultured cells, target genes in different lineages, and the intrinsic abilities of hematopoietic stem cells. In the first study, we found that T cell and NK cell genes were repressed in a gradient fashion by increasing levels of PU.1 in cytokine-dependent ex vivo myeloid and pro-B cultured cells. In the second study, we found that PU.1 activated the myeloid/B cell gene encoding FcgammaRIIb and repressed the invariant natural killer/T cell (iNKT) gene encoding PLZF through conserved regulatory elements and chromatin remodeling. In the third study, we found that fetal liver cells with reduced levels of PU.1 have increased self-renewal capacity. We also found that while fetal liver cells with reduced levels of PU.1 were severely impaired in the ability to engraft and reconstitute hematopoiesis, neonatal spleen cells were able to engraft. Interestingly, they gave rise to a large B cell population with increased expression of IL-7Ralpha and a higher frequency of B-1 versus B-2 cells as well as cells that coexpressed myeloid and T cell lineage markers. In summary, this thesis project further defined the role of PU.1 in hematopoiesis. PU.1 is vital for the proper development of the myeloid and B cell lineages due to its function in hematopoietic stem cell differentiation, activation of myeloid and B cell genes, and the concomitant repression of genes from alternate lineages, such as T cell genes. This highlights the importance of lineage-appropriate gene expression in maintaining lineage identity and hematopoietic development. |
