Trancriptomic analysis of hematopoietic stem cells (HSCs) from mice expressing wild-type, Knock-In, knock-down, knock-out and 36N SNP variants of GFI1.
GFI1 is a transcriptional repressor that plays an essential role in HSCs development, lymphoid and myeloid differentiation and Acute Myeloid Leukemia (AML) pathogenesis. Low expression of Gfi1 leads to poor prognosis in AML patients and is associated with less overall survival in murine AML models. In the current study, we show that mice with a low level or loss of GFI1 expression resulted in significantly fewer HSCs compared to normal GFI1 expression. We also show that, in a competitive transplantation setup involving cells expressing low and normal GFI1 levels, HSCs with a low level of GFI1 expression reconstituted the bone marrow (BM) including the HSCs, progenitors and differentiated cells. In contrast, HSCs with the complete loss of GFI1 failed to regenerate BM in competition with cells expressing normal GFI1 levels. To further investigate the molecular changes, we performed RNAseq analysis in HSCs derived from wildtype (GFI1-KI), low-level (GFI1-KD), loss (Gfi1-KO) and Gfi1 mutant (GFI1-36N) mice. In the pathway analysis, we observed a significant upregulation of cell-cycle-related pathways in HSCs derived from mice expressing low-level and mutant variant (36N) of GFI1., Strikingly, these pathways are significantly downregulated in the HSCs of GFI1-KO mice. Overall design: In the current study, we performed RNAseq analysis with HSCs isolated from five different mouse models. Gfi1-WT mice that express murine Gfi1, GFI1- KI mice, that express human GFI1 in murine locus, GFI1-KD mice that express 10-20% of human GFI1 gene, Gfi1-KO mice that show the complete absence of Gfi1 and GFI1-36N mice expressing mutant human GFI1-36N variant in the murine locus. The HSCs were FACS sorted and RNA was isolated immediately.