LRF Is Indispensable for Hematopoietic Stem Cell Function Via Blocking Notch1-Mediated T Cell-Instructive Signals in the Bone Marrow Niche.

Abstract 81

Hematopoietic stem cells (HSC) have the ability to self-renew and give rise to all hematopoietic lineage cells. Understanding signals that regulate the balance between self-renewal and differentiation of HSCs is an important issue in stem cell biology as well as regenerative medicine. Notch signals are critical regulators of the lymphoid lineage fate, but their role in adult HSC function is currently under debate. We explored the role of the LRF (Leukemia/Lymphoma Related Factor), a Notch repressor (also known as Zbtb7a, pokemon, OCZF and FBI-1) in HSC function, as it plays key roles in embryonic development, oncogenesis, and hematopoiesis. Conditional inactivation of the LRF gene in mouse HSCs (LRF^F/FMx1-Cre mice) led to the development of CD4/CD8 DP (double positive) T-cells at the expense of B-cell development in the bone marrow (BM) in a Notch-dependent manner. Absolute numbers of the most primitive HSCs (LT-HSCs), defined as CD150⁺CD48⁻Flt3⁻Vcam-1⁺IL7Rα⁻LSK (Lin⁻Sca1⁺c-Kit⁺), were significantly reduced, while lymphoid-biased multi-potential progenitors (LMPPs: CD150⁻CD48⁺Flt3⁺Vcam-1^+/−IL7Rα⁻LSK) and common lymphoid progenitors (CLPs: Lin⁻CD150⁻CD48⁺Flt3⁺Vcam-1⁻IL7Rα⁺) were barely detectable in LRF^F/FMx1-Cre mice one month after pIpC injection. Enhanced T cell development and concomitant loss of B cell development was also seen in LRF^−/− fetal liver (FL). Lin⁻IL7Rα⁺c-Kit⁺PIR⁺ (Paired Immunoglobulin-like receptors) T cell precursors were significantly increased in LRF^−/− FL, indicating that Notch-mediated aberrant lymphoid fate determination also takes place during fetal hematopoiesis. To address which Notch gene(s) are targeted by LRF, we studied the HSC/progenitor population of conditional LRF knockout (LRF^F/FMx1-Cre) as well as LRF/Notch1 double conditional knockout mice (LRF^F/FNotch1^F/FMx1-Cre). In the absence of Notch1, normal B cell development was restored in LRF^F/FMx1-Cre mice. Reduction of LT-HSCs in LRF^F/FMx1-Cre resulted from high Notch1 activity, as loss of Notch1 rescued LT-HSC numbers, suggesting that LRF functions to maintain HSCs and normal lymphoid fate by blocking Notch1. HSCs in active cell cycle are sensitive to 5-fluoro-uracil (5-FU) treatment, which causes remaining dormant HSCs to be recruited into the cell cycle to rapidly produce new cells and to quickly re-establish the hematopoietic system. To examine the self-renewal capacity of LRF deficient LT-HSC, LRF^F/FMx1-Cre mice were treated with 5-FU after pIpC injection and the recovery of LT-HSC numbers examined. While control LT-HSC numbers recovered to pretreatment levels 3 wk after 5-FU treatment, levels in LRF^F/FMx1-Cre mice remained low, accompanied by DP T cell development in the BM. Furthermore, after 5-FU treatment, LT-HSC numbers of LRF^F/FNotch1^F/FMx1-Cre were compatible to those of control and LRF^F/FMx1-Cre mice, indicating that lack of self-renewal capacity in LRF deficient LT-HSCs was due to excessive differentiation toward T cells caused by Notch1. In support of this idea, when mice were given 5-FU weekly as a challenge to assess their HSC function in vivo, the survival percentage in LRF^F/FMx1-Cre mice was much lower than in controls (0% versus 50% in 1 month, P <0.0001) and that of LRF^F/FNotch1^F/FMx1-Cre mice was compatible to controls. Serial bone marrow transplant experiments further demonstrated functional defects of LRF deficient HSCs, as they failed to reconstitute the hematopoietic system in secondary recipients. Microarray analysis and subsequent Gene Set Enrichment Analysis demonstrated upregulation of genes that were enriched in progenitor compartments. Since LRF can act as a transcriptional repressor, mRNA levels of Notch receptors and Notch ligands were examined using the same data set. A Notch target gene Hes1, but not Notch1 itself, was upregulated, and increased levels of Hes1 was also confirmed by real-time q-PCR in FACS-sorted LT-HSCs, as well as in 10.5 d.p.c whole embryos. These data suggest that LRF does not transcriptionally regulate Notch1, as LRF loss led to Notch1 target gene activation at the LT-HSC level without affecting Notch1 mRNA. Our genetic studies clearly indicate that LRF is indispensable for the maintenance of the HSC pool by repressing T cell-instructive signals mediated by Notch1 in the BM niche. Our findings shed new light on the regulatory mechanisms underlying the balance between HSC self-renewal and differentiation.