Role of the Transcription Factor LYL-1 in the Adult and Fetal Hematopoietic Stem Cells.

The hematopoietic stem cells (HSC) have the ability to self-renew and to give rise to all blood lineages. These processes occur via a hierarchy of progenitors with progressively more limited differentiation and self-renewal potential and are orchestrated by specialized protein such as transcription factors. LYL-1 protein contains a basic helix-loop-helix DNA binding motif also found in several proteins involved in the control of cellular proliferation and differentiation such as SCL/TAL-1. As LYL-1 shares an 80% homology at the protein level with SCL/TAL-1, we wanted to determine the function of LYL-1 in hematopoiesis and particularly on HSC. For this study, we used knock in lyl-1^−/− mice in which exon 4 was replaced by LacZ/Neo cassette. Lyl^−/− mice are viable and have normal blood cell counts as well as a normal marrow cellularity. In addition, using a hematopoietic colony forming cells (CFCs) assay, no significant difference was seen in the myeloid CFCs of either lyl-1^−/− or lyl-1^+/+ BM and FL cells except a 2-fold increase in the absolute number of BFU-E in lyl-1^−/− FL as compared to lyl-1^+/+ FL. We analyzed more primitive progenitors in details because using Fluorecein Di-beta Galactopyranoside (FDG)-staining assay, we showed that lyl-1 is mainly expressed in primitive Lin⁻ Sca-1⁺ c-Kit⁺ cells (LSK) cells from either BM or FL (91 ± 7% and 78 ± 5% of FDG positive cells in lyl-1^−/− BM and FL LSK cells, respectively). In addition, analysis of lyl-1^−/− and lyl-1^+/+ cells revealed a 1.8-fold and 2-fold decrease in the percentage of primitive LSK in BM and FL, respectively, as compared to wild type cells. Furthermore, using the Hoechst 33342 efflux assay, we noticed a significant decrease in the absolute number of more primitive LSK-SP (side population) cells in lyl-1^−/− BM as compared to lyl-1^+/+ BM cells (52800 ± 5412 cells/femur versus 91080 ± 8475 cells/femur, respectively) suggesting an important role of LYL-1 in the HSC function. In order to confirm this hypothesis, in vivo assays were performed. We observed a 1.5-fold decrease in the lyl-1^−/− BM and FL day 12 CFU-S content as compared to lyl-1^+/+ cells. Adoptive transfer experiments were subsequently performed using lethally irradiated Ly5.1 mice. Data showed that lyl-1^−/− cells from either BM or FL displayed a hematopoietic reconstitution defect in competitive repopulation assays. Indeed, Ly5.1 recipients were injected with a mixture of 5x10⁶ (5:1), 10⁶ (1:1) or 0.5x10⁶ (0.5:1) lyl-1^−/− or lyl-1^+/+ Ly5.2 expressing cells and 10⁶ competitive BM Ly5.1 expressing cells. All hosts engrafted with lyl-1^−/− BM cells shown a significant reduced levels of chimerism (% of circulating Ly5.2⁺ cells) as compared to hosts engrafted with lyl-1^+/+ BM donors (4.3 ± 2.8% (5:1); 7.5 ± 5.5% (1:1); 0.6 ± 0.3% (0.5:1) in lyl-1^−/− BM cells versus 66 ± 8% (5:1); 52 ± 9% (1:1); 53 ± 10% (0.5:1) in lyl-1^+/+ BM cells) and similar difference was observed with FL donors (45 ± 2% (5:1); 25 ± 5% (1:1); 11 ± 5% (0.5:1) in lyl-1^−/− FL cells versus 83 ± 1% (5:1); 70 ± 3% (1:1); 53 ± 6% (0.5:1) in lyl-1^+/+ FL cells). This altered defect in HSC was also confirmed using LTC-IC in vitro experiments. Altogether, our results demonstrate an important role of the transcription factor LYL-1 on the maintenance of HSC properties.