Recent studies have identified a number of transcriptional regulators, including E2A, early B-cell factor 1 (EBF1), FOXO1, and paired box gene 5 (PAX5), that promote early B-cell development. However, how this ensemble of regulators mechanistically promotes B-cell fate remains poorly understood. Here we demonstrate that B-cell development in FOXO1-deficient mice is arrested in the common lymphoid progenitor (CLP) LY6D + cell stage. We demonstrate that this phenotype closely resembles the arrest in B-cell development observed in EBF1-deficient mice. Consistent with these observations, we find that the transcription signatures of FOXO1-and EBF1-deficient LY6D + progenitors are strikingly similar, indicating a common set of target genes. Furthermore, we found that depletion of EBF1 expression in LY6D + CLPs severely affects FOXO1 mRNA abundance, whereas depletion of FOXO1 activity in LY6D + CLPs ablates EBF1 transcript levels. We generated a global regulatory network from EBF1 and FOXO1 genome-wide transcription factor occupancy and transcription signatures derived from EBF1-and FOXO1-deficient CLPs. This analysis reveals that EBF1 and FOXO1 act in a positive feedback circuitry to promote and stabilize specification to the B-cell lineage.

IntroductionThe development of B-lymphoid cells from hematopoietic stem cells in the bone marrow (BM) is achieved through a stepwise process where multipotent cells gradually lose lineage potentials before committing to B-lineage cell fate. The precise mechanism for this is still under investigation, but several factors with key roles in this process have been identified. One of these is the cytokine IL-7 because in the absence of either the cytokine 1 or the ␣-chain of the receptor, 2 B-lymphocyte development is severely impaired and only low numbers of B-lineage progenitors can be found in the BM of adult mice. Even though T-cell development is impaired in the absence of functional IL-7 signaling, the reduction in T-lymphocyte progenitors can be rescued by ectopic expression of Bcl-2. [3][4][5] Expression of this antiapoptotic factor is, however, insufficient to rescue B-lymphocyte development, 4,6 suggesting that although IL-7 is permissive for T-cell progenitors, this cytokine might play a more unique role in B-lineage differentiation. Although the block in development of committed CD19 ϩ B-lineage progenitors is obvious, the function of IL-7 signaling in early lymphoid cells is more controversial. Although the early thymic progenitors essentially lack expression of IL-7R, 7 the common lymphoid progenitor (CLP) compartment 8 as well as a small fraction of the lymphoidprimed multipotent progenitor (LMPP) compartment 9 express the receptor. Initial investigations of IL-7R-deficient mice suggested that the CLP compartment developed normally, whereas the development of the progenitor B-cell compartment was dramatically impaired. 10 The obvious disadvantage of studying early lymphoid compartments using IL-7R-deficient mice motivated further investigations of the CLP compartment using IL-7-deficient mice where the conventional surface markers for the identification of early populations can be used. These experiments indicated that the addition of IL-7 to CLPs from IL-7-deficient mice was not sufficient to allow for proper B-cell development in vitro, suggesting that IL-7 is critical for the development of B-lineage competent CLPs. 11,12 The B-lineage potential could be partially rescued by ectopic expression of the transcription factor Ebf-1, present at reduced levels in CLPs from IL-7-deficient mice compared with their wild-type (wt) counterparts. [11][12][13] Expression of this transcription factor in the CLP compartment is of highest relevance for B-lymphocyte commitment because targeted disruption of the Ebf-1 gene results in that the transcription of B-lineageassociated genes is dramatically reduced. 14 The Ebf-1 gene also has been reported to be a direct target for Stat-5, one of the critical signal transducers downstream of the IL-7 receptor in B-cell development, 13,15 creating a direct link between IL-7 signaling and Ebf-1 expression. 16 Although most of these findings would indicate that IL-7 is critical for normal expression of Ebf-1 and preserved B-lineage potential in B-cell progenitors, 13,15 it was ...

Key Points Ebf1 regulates DNA repair in a dose-dependent manner. Combined heterozygote loss of Ebf1 and Pax5 predisposes for leukemia development.

To better understand the process of B-lymphocyte lineage restriction, we have investigated molecular and functional properties in early Blineage cells from Pax-5-deficient animals crossed to a B-lineage-restricted reporter mouse, allowing us to identify B-lineage-specified progenitors independently of conventional surface markers. Pax-5 deficiency resulted in a dramatic increase in the frequency of specified progenitor B-cells marked by expression of a λ5 (Igll1) promotercontrolled reporter gene. Gene expression analysis of ex vivo isolated progenitor cells revealed that Pax-5 deficiency has a minor impact on B-cell specification. However, single-cell in vitro differentiation analysis of ex vivo isolated cells revealed that specified Blineage progenitors still displayed a high degree of plasticity for development into NK or T lineage cells. In contrast, we were unable to detect any major changes in myeloid lineage potential in specified Pax-5-deficient cells. By comparison of gene expression patterns in ex vivo isolated Pax-5-and Ebf-1-deficient progenitors, it was possible to identify a set of B-cell-restricted genes dependent on Ebf-1 but not Pax-5, supporting the idea that B-cell specification and commitment is controlled by distinct regulatory networks.

Ungerbäck et al. show that transcription factors Ebf1 and Pax5 act in a coordinated, dose-dependent manner to preserve B-lineage cell fate. Combined heterozygous loss of both transcription factors results in increased T cell lineage skewing in B cell progenitors.