Transcription factor EBF restricts alternative lineage options and promotes B cell fate commitment independently of Pax5.

Alternative lineage restriction and B cell fate commitment require the transcription factor Pax5, but the function of early B cell factor (EBF) in these processes remains mostly unexplored. Here we show that in the absence of EBF, 'expandable' and clonal lymphoid progenitor cells retained considerable myeloid potential. Conversely, ectopic expression of EBF in multipotential progenitor cells directed B cell generation at the expense of myeloid cell fates. EBF induced Pax5 and antagonized expression of genes encoding the transcription factors C/EBPalpha, PU.1 and Id2. Notably, sustained expression of EBF in Pax5-/- hematopoietic progenitor cells was sufficient to block their myeloid and T lineage potential in vivo. Furthermore, in Pax5-/- pro-B cells, higher EBF expression repressed alternative lineage genes. Thus, EBF can restrict alternative lineage 'choice' and promote commitment to the B cell fate independently of Pax5.

Assembling a gene regulatory network for specification of the B cell fate.

The generation of B lymphocyte precursors is dependent on the combinatorial action of the transcription factors PU.1, Ikaros, E2A, EBF, and Pax-5. Loss of PU.1 results in a severe reduction in Flk2+, IL-7R+ lymphoid progenitors as well as impaired expression of EBF and Pax-5. Restoration of EBF expression facilitates rapid generation of pro-B cells from PU.1-/- progenitors. Molecular analysis suggests that PU.1 directly participates in regulation of the EBF gene. Although PU.1 is dispensable for expression of most early B lineage genes, it is required for CD45R/B220. Using EBF-/- mutant progenitors, we show that EBF induces Pax-5 and the early program of B lineage gene expression. Importantly, Pax-5 does not rescue B cell development from either PU.1-/- or EBF-/- progenitors. Pax-5 expression and function are contingent on EBF. Based on these results, we propose a hierarchical regulatory network for specification and commitment to the B cell fate.

Gene regulatory networks directing myeloid and lymphoid cell fates within the immune system.

Considerable progress is being achieved in the analysis of gene regulatory networks that direct cell fate decisions within the hematopoietic system. In addition to transcription factors that are pivotal for cell fate specification and commitment, recent evidence suggests the involvement of microRNAs. In this review we attempt to integrate these two types of regulatory components into circuits that dictate cell fate choices leading to the generation of innate as well as adaptive immune cells. The developmental circuits are placed in the context of a revised scheme for hematopoiesis that suggests that both the innate (myeloid) and adaptive (lymphoid) lineages of the immune system arise from a common progenitor.

Multilineage transcriptional priming and determination of alternate hematopoietic cell fates.

Hematopoietic stem cells and their progenitors exhibit multilineage patterns of gene expression. Molecular mechanisms underlying the generation and refinement of these patterns during cell fate determination remain unexplored because of the absence of suitable experimental systems. Using PU.1(-/-) progenitors, we demonstrate that at subthreshold levels, this Ets transcription factor regulates a mixed pattern (macrophage/neutrophil) of gene expression within individual myeloid progenitors. Increased PU.1 levels refine the pattern and promote macrophage differentiation by modulating a novel regulatory circuit comprised of counter antagonistic repressors, Egr-1,2/Nab-2 and Gfi-1. Egr-1 and Egr-2 function redundantly to activate macrophage genes and to repress the neutrophil program. These results are used to assemble and mathematically model a gene regulatory network that exhibits both graded and bistable behaviors and accounts for the onset and resolution of mixed lineage patterns during cell fate determination.

IRF-4,8 orchestrate the pre-B-to-B transition in lymphocyte development.

B-lymphocyte development involves sequential DNA rearrangements of immunoglobulin (Ig) heavy (mu) and light (kappa, lambda) chain loci and is dependent on transient expression of mu containing pre-antigen receptor complexes (pre-BCR). To date, genetic analysis has not identified transcription factors that coordinate the pre-B-to-B transition. We demonstrate that the related interferon regulatory factors IRF-4 (Pip) and IRF-8 (ICSBP) are required for Ig light but not heavy-chain gene rearrangement. In the absence of these transcription factors, B-cell development is arrested at the cycling pre-B-cell stage and the mutant cells fail to down-regulate the pre-BCR. On the basis of molecular analysis, we propose that IRF-4,8 function as a genetic switch to down-regulate surrogate light-chain gene expression and induce conventional light-chain gene transcription and rearrangement.

Cooperative and antagonistic interplay between PU.1 and GATA-2 in the specification of myeloid cell fates.

PU.1 and GATA transcription factors appear to antagonize each other's function in the development of distinct lineages of the hematopoietic system. In contrast, we demonstrate that PU.1, like GATA-2, is essential for the generation of mast cells. PU.1-/- hematopoietic progenitors can be propagated in IL-3 and differentiate into mast cells or macrophages upon restoration of PU.1 activity. Using these progenitors and a conditionally activatable PU.1 protein, we show that PU.1 can negatively regulate expression of the GATA-2 gene. In the absence of GATA-2, PU.1 promotes macrophage but not mast cell differentiation. Reexpression of GATA-2 in such progenitors enables the generation of mast cells. We propose a developmental model in which cooperative function or antagonistic crossregulation by PU.1 of GATA-2 promotes distinct myeloid cell fates.