The correct production of eight discrete blood cell lineages in the mammal is dependent on a cell hierarchy composed of cells with increasingly committed fates. The foundation of the hierarchy resides in a population of uncommitted stem cells which must balance decisions to self-renew, commit to differentiation and proliferate. The molecular regulatory mechanisms which control these decisions underlie the entire hematopoietic system. While much is known about regulatory pathways that influence more mature members of the hematopoietic hierarchy, almost no information is available concerning the regulatory mechanisms which function in the most primitive stem cells. Gene products clearly influence the behavior of stem cells however there has not been a systematic effort to screen for and identify regulatory gene products directly by their activities in stem cells. We propose such an approach. Our strategy takes advantage of and integrates several technologies previously applied to hematopoietic studies. Specifically, a large population of gene products enriched in those preferentially expressed in stem cells will be retrovirally-transduced into isolated fetal liver stem cells. These cells will be assayed in vitro and in vivo for alterations in properties such as self renewal, commitment and proliferative potential. Additional studies are proposed which focus on the identification of stem cell regulators which require dimerization for functional activation. Finally, the hematopoietic role of a novel, transcriptional regulator in the Polycomb protein family will be analyzed using gain-of-function and loss-of-function in vivo approaches. For these experiments conditional transgenic and """"""""knockout"""""""" strategies will be employed. Collectively, the proposed studies should complement other, more traditional sequence and expression based efforts to identify stem cell regulators.
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