LRF/Pokemon Plays a Pivotal Role in B Versus T Lymphoid Lineage Fate Decision at the Early Lymphoid Progenitor Stage by Opposing Notch1 Signaling

LRF (Leukaemia/Lymphoma Related Factor, formerly described as Pokemon, FBI-1 and OCZF, encoded by the Zbtb7a gene) is a transcriptional repressor that belongs to the POK (POZ/BTB and Krüppel) protein family. We recently reported that LRF is a proto-oncogene, which is highly expressed in Non-Hodgkin’s Lymphoma tissues (Nature. 433, 278–85). To elucidate its function in fetal and adult lymphopoiesis, we analyzed LRF knockout mice (Zbtb7a−/−) and the conditional knockout mutants (Zbtb7a flox/−Mx-1cre+), respectively. Zbtb7a −/− mice are embryonic lethal due to severe anemia around 16.5 d.p.c. Absolute number of mature B cells was markedly decreased in 15.5 d.p.c Zbtb7a−/− fetal livers (FL), while total number of the earliest immature B cells (Lin-AA4.1+CD19−B220+) was comparable to wild type (WT) littermates. Flow-Sorted Zbtb7a−/− FL Hematopoietic Stem Cells (FL-HSCs) did not give rise to ProB cells in vitro in an OP9 cell culture system. Furthermore, competitive repopulation assay suggested that the defect in B cell development in Zbtb7a−/− FL was cell-autonomous. Conditional inactivation of LRF in adult mice resulted in a significant decrease of B220+ B cells in the peripheral blood (PB). Absolute numbers of both ProB and PreB cells in the BM were drastically reduced in Zbtb7a flox/−Mx-1cre+ mice after pIpC injections, while PreProB cells were rather accumulated. Zbtb7a flox/−Mx-1cre+ PreProB cells did not give rise to ProB cells in vitro in OP9 cell culture. Unexpectedly, Zbtb7a flox/−Mx-1cre+ PreProB cells ectopically expressed T cell genes (e.g. pTCRα, Notch1, Notch3, Hes1, Gata3, TCF1), while they lacked B-cell specific gene expression (e.g. E2A, Ebf1, Pax5, Rag, VpreB1). In agreement with this finding, Zbtb7a flox/−Mx-1cre+ PreProB cells efficiently differentiated into DP-T cells upon 6 days of culture on OP9-DL1 cells, which overexpress Notch1 ligand Delta-like1. We did not observe a gross defect in the T cell compartment in PB and Thymus of Zbtb7a flox/−Mx-1cre+ mice. However, we observed an accumulation of CD4/8 double positive (DP) T cells in their BM. DP-T cells consisted of nearly 30% of the BM mononuclear cells (MNCs) in Zbtb7a flox/−Mx-1cre+ mice. Since the phenotype of LRF conditional knockout mice is reminiscent of that of ICN1 (Intracellular domain of Notch1) overexpression in the mouse BM, we hypothesized that LRF could oppose Notch1 signaling pathway at the early lymphoid progenitor stage. We found that pTCRα, a Notch1 target gene, is highly up-regulated in Zbtb7a flox/−Mx-1cre+ CLPs and that LRF transcriptionally represses mouse pTCRα promoter activity. Our finding strongly indicates that LRF loss at the early lymphoid progenitor stage causes aberrant de-repression of T-cell specific genes, which results in the block of B cell development and generation of T cells in the BM. We therefore propose that LRF is essential in instructing the early lymphoid progenitors into B cell lineage by repressing T cell-instructive signal produced by Notch.