Dose-dependent role of Gfi1 in murine hematopoietic stem cell self-renewal and differentiation

Abstract

21Gfi1 (Growth factor independence 1) is a transcription factor that influences the stem cell 22 capacity of hematopoietic stem cells (HSCs) as well as their differentiation into the myeloid 23 and lymphoid lineage. Loss of Gfi1 impedes the repopulation capacity of HSCs and leads to a 24 block in granulocyte generation causing severe neutropenia and monocytosis. Competitive 25 transplantation assays showed that Gfi1-deficient cells were not able to reconstitute myeloid 26 and lymphoid hematopoiesis in competition with Gfi1-wildtype (GFI1-36S) cells. Low Gfi1 27 levels (GFI1-knockdown = GFI1-KD) in blasts of myelodysplastic neoplasms, acute and 28 chronic myeloid leukemia patients are associated with poor patient survival. To understand 29 how reduced levels or loss of Gfi1 contribute to hematopoiesis, we analyzed the effect of 30 GFI1-KD and Gfi1-KO on HSCs and more mature cell types in mice. GFI1-KD and Gfi1-KO 31 led to strong decrease in HSC numbers, while the numbers of early progenitors (Lin -32 Sca1 + cKit + cells) were slightly increased. Competitive transplantation assays showed that 33 GFI1-KD and Gfi1-KO HSCs can still engraft and expand, but they cannot contribute to 34 myeloid and lymphoid differentiation. 35 36 42 (granulocyte-macrophage progenitors) and CLPs (common lymphoid progenitors), while it is 43 absent in CMPs (common myeloid progenitors) and MEPs (megakaryocyte-erythroid 44 progenitors) (6). Gfi1-deficient mice are characterized by an accumulation of monocytic cells 45 and absence of granulocytes, leading to severe neutropenia and monocytosis (7, 8). Recently 46 it has been implicated that reduced levels of Gfi1 are associated with an inferior prognosis for 47 human MPN (myeloproliferative neoplasm), CML (chronic myeloid leukemia) and AML 48 (acute myeloid leukemia) patients and with an acceleration of AML development in mice (9-49 11). These studies also showed that Gfi1 regulates expression of certain oncogenes, apoptotic 50 pathways and possibly metabolic functions in a dose-dependent manner. However, it has also 51 been postulated that loss of Gfi1 negatively influences the repopulation capacity of HSCs (6, 52 12). 53We were now interested whether low levels and loss of Gfi1 indeed negatively affect the stem 54 cell and differentiation capacity of HSCs and how the seemingly contradictory findings 55 between reduced self-renewal capacity and the dose-dependent role of Gfi1 function in 56 myeloid pathogenesis could be reconciled. For the analysis we made use of Gfi1-knockout 57 (KO) mice, a mouse strain with a complete loss of Gfi1, and GFI1-knockdown (KD) mice, a 58 mouse strain in which we cloned the human GFI1 sequence into the murine Gfi1 gene locus 59 together with a Neo cassette in the opposite direction of transcription, leading to GFI1 60 expression of only 10-15% of wild-type levels (10). We show here that GFI1-KD and Gfi1-61 KO mice contain a higher number of hematopoietic progenitors (LSK cells, Lin -Sca1 + c-Kit + ), 62 but HSC (Lin -Sca1 + c-Kit + CD150 + CD48 -) numbers are ...