Determination of the Role and Mechanism of the Transcription Factor NF-Y in Murine Hematopoietic Stem Cell Biology.

Previous studies from our and other laboratories have demonstrated that the trimeric transcription factor NF-Y is a potent inducer of many of the genes implicated in hematopoietic stem cell (HSC) self-renewal, suggesting that NF-Y functions as a dominant regulator of genes controlling the balance between self-renewal and differentiation of stem cells. Furthermore, over-expression of NF-Ya, the regulatory subunit of NF-Y, was shown to increase HSC potency in vivo through increased expression of a whole series of genes playing central roles in stem cell function including HoxB4 and Notch-1. The importance of the NF-Y transcription factor for mammalian development is further highlighted by a study demonstrating that a loss of function mutation of NF-Ya in mice, leads to lethality before day E8.5 (Bhattarcharya A. et al. 2003). Therefore, it can be reasoned that the NF-Y transcription factor might act as a master gene among the network of genes involved in early development regulating self-renewal and differentiation of (hematopoietic) stem cells. A concept in cancer biology, well-established in chronic myelogenous leukemia (CML), is that a rare population of cancer stem cells (CSSs) exists that is capable of extensive self-renewal, while most tumor cells have a limited proliferative capacity. Many studies suggest that the similar behavior of HSCs and CSSs are due to similar, yet not identical, molecular mechanisms determining whether a stem cell self-renews or differentiates. Knowledge about the regulatory mechanisms underlying cellular NF-Y abundance and activity and thereby NF-Y-mediated fate decision of SCs would open an elegant way to manipulate SCs, including leukemic stem cells (LSC), for therapeutic use. In this study, we have determined NFYa to have an essential role in murine HSC biology. To circumvent embryonic lethality, we generated bone marrow (BM) chimeric mice in which the deletion of functional NF-Ya can be induced selectively in the hematopoietic system. The analysis of lineage committed cells of the BM, spleen and thymus ten weeks after the disruption of the NF-Ya gene revealed an essential role for NF-Y activity in the hematopoietic system. Furthermore, BM cells from wild type, heterozygous and NF-Ya mutant BM chimera were subjected to colony formation assays, clearly demonstrating the indispensability of NF-Y function for hematopoietic stem and precursor cells. At that time not a single colony deficient for NF-Ya could be found, highlighting the absolute necessity of NF-Y activity for HPCs and HSCs. To explain these deleterious defects mechanistically, the role of NF-Y in regulating potential target genes that in turn control hematopoietic stem cell behavior is comprehensively addressed. Our approach is to ectopically express these downstream genes, such as HoxB4, Notch-1, Bmi-1 and Lef-1 in vivo and subsequently delete NF-Y activity. The results from these assays reveal information about the mechanistic interplay and the position within different pathways of these proteins in HSC behavior. Since the loss of NF-Y has lethal consequences for normal HSCs we are currently testing the effects of NF-Y deletion on LSCs in vivo using an established mouse model for CML. If LSCs and normal HSCs share a common machinery of fate-regulation, it is expected that NF-Ya is essential for LSC survival. If it turns out though, that LSCs and HSCs can be discriminated upon their dependence on NF-Y activity, this would harbor tremendous therapeutic possibilities with medical relevance extending into cancer therapy.