LRF Regulates Self-Renewal of Hematopoietic Stem Cells by Blocking Notch1-Mediated T Cell Differentiation

The proto-oncogene LRF, encoded by the Zbtb7a gene, is a transcriptional repressor that belongs to the POK (POZ/BTB and KrŸppel) protein family. Along with its oncogenic property, recent evidence has shown that POK proteins play distinct roles in hematopoiesis and immune system development. Conditional inactivation of the LRF gene in mouse hematopoietic stem cells (HSCs) results in the development of CD4/8 double positive (DP) T cells in bone marrow (BM) at the expense of B cell development (Maeda et al. Science 2007). While LRF acts as a master regulator of B versus T lymphoid lineage fate decision by suppressing Notch-mediated signals, it is unclear as to

1. which Notch genes LRF targets and

2. whether LRF is required for the maintenance of HSCs per se.

To address these questions, we analyzed HSC/progenitor population of conditional LRF knockout mice (LRF^F/FMx1-Cre) as well as LRF/Notch1 double conditional knockout mice (LRF^F/FNotch1^F/FMx1-Cre). In the absence of Notch1, LRF deficient HSCs/lymphoid progenitors (LRF^F/FNotch1^F/FMx1-Cre) could successfully give rise to early B cells (Pro B, Pre B and immature B). There were no abnormal DP-T cells seen in the BM, suggesting that LRF primarily targets Notch1 at the HSC/progenitor stages to maintain normal lymphoid development. However the loss of the LRF gene did not rescue the phenotype of Notch1^F/FMx1-Cre mice (Radtke et al. Immunity 1999). Immature B cell development in the thymus was still observed in LRF^F/FNotch1^F/FMx1-Cre mice, suggesting that LRF acts genetically upstream of Notch1 during the early lymphocyte development. Notably, LRF^F/FNotch1^F/FMx1-Cre mice still exhibit a block of terminal erythroid differentiation and macrocytic anemia as seen in LRF^F/FMx1-Cre mice. Thus, LRF is required for erythropoiesis via Notch-independent mechanisms. To further identify distinct HSC/progenitor compartments, we performed multicolor-FACS analysis utilizing antibodies for SLAM family members (CD41, CD48 and CD150), c-Kit, Sca-1, Flt3, IL7R-α, Vcam-1 and lineage markers (Lin). Remarkably, no Flt3 positive HSC/progenitors were observed in LRF^F/FMx1-Cre mice. While IL7R-α+ T cell precursors (IL7Rα+Lin-Sca1+c-Kit+Flt3-), which were previously reported as common lymphoid progenitors (Maeda et al. Science 2007), existed abundantly. Absolute numbers of the long-term HSCs (LT-HSCs), defined as CD150+CD48-Flt3-Vcam-1+IL7Rα-LSK (Lin-Sca1+c-Kit+), were significantly reduced in LRF^F/FMx1-Cre mice one month after pIpC injection. At the same time, CD150+CD48high+Flt3-Vcam-1-IL7Rα-LSK cells, which are likely T-committed lymphoid precursors, are increased in LRF^F/FMx1-Cre mice. To investigate the presence of a population of quiescent HSC/progenitors, we treated LRF^F/FMx1-Cre mice with 5-fluorouracil (5-FU), a S phase-specific cytotoxic chemotherapeutic agent, and examined recovery of HSCs in BM. LT-HSCs in LRF^F/FMx1-Cre mice did not repopulate as many as their counterpart one month after 5-FU treatment. Our data indicates that LRF deficient HSCs are unable to maintain its quiescent status and are on the state of cell differentiation toward T cells due to the high Notch activity. In fact, loss of the Notch1 gene partially rescued reduced LT-HSCs numbers seen in LRF^F/FMx1-Cre mice.