Research Description:

PU.1'S Roles in Myeloid Development and Adult Stem Cell Differentiation

Hematopoiesis is the regulated development of pluripotential stem cells into mature blood cell types. It is through this process that mature monocytes, granulocytes, lymphocytes, megakaryocytes, and erythrocytes are regenerated throughout development and adulthood. Regulation of this process is under the control of a cascade of intercellular and extracellular signals, one being the transcription factor PU.1. Previous in vitro studies have shown PU.1 to contain three functional domains necessary for macrophage development. PU.1 also seems to influence the developmental fate of adult stem cells as PU.1 -/- HSC regenerate liver more robustly than wild type HSC. This indicates that PU.1 -/- HSC may have a proclivity to contribute to other non-hematopoietic lineages.

My project is thus to examine more specifically PU.1’s role in HSC differentiation. The first aim of the project is to determine the function’s for each of PU.1’s domains in vivo. It has already been shown that PU.1 -/- mutants die at a late gestational stage. Primitive macrophages do develop, but functional macrophage development is completely blocked. I will thus examine how mutations to only specific acidic, glutamine-rich, and ETS domains affect differentiation. This will be done by creating a series of PU.1 knock-in mice containing the one of the three mutated domains. After creation of the transgeneic mouse, I will use southern blots, microarrays, and FACS to look for various hematopoietic lineages and if they were affected.

The second aim of the project is to examine PU.1’s role in adult HSC differentiation and whether or not PU.1 contributes to non-hematopoietic lineages. This will be done by creating an anti-PU.1 ribozyme to effectively knock out the protein in adult mice. After knocking out the protein, hematopoietic stem cells and their ability to become other non-hematopoietic cell types will be monitored.