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. Author manuscript; available in PMC: 2012 Apr 1.
Published in final edited form as: Curr Opin Immunol. 2011 Jan 12;23(2):163–170. doi: 10.1016/j.coi.2010.11.014

Molecular Resolution of the B Cell Landscape

Patricia M Santos 1, Lisa Borghesi 1,1
PMCID: PMC3073704  NIHMSID: NIHMS264999  PMID: 21236654

Summary

The progression of hematopoietic stem cells (HSCs) to the B lymphocyte lineage requires that uncommitted progenitors successfully negotiate the transition from multipotency to unipotency, including the loss of self-renewal potential. Previous work identified essential transcription factors that mediate B lineage development. Major advances build on this knowledge and reveal coordinated changes in gene expression occurring within single cells at sequential stages in the B cell differentiation pathway. Recent studies on epigenetic mechanisms also provide a framework within which transcription factor activity, chromatin modifications, and gene expression patterns can be viewed at hierarchical levels to link genotype and phenotype.

Introduction

Hematopoiesis is a key model system for understanding the mechanisms that control tissue regeneration, developmental plasticity, and lineage fate decisions. This is a scientifically exciting area because the basic processes that occur during hematopoiesis are also fundamental to embryogenesis and tissue repair/regeneration. As such knowledge about the molecular mechanisms that control hematopoiesis have broad biological implications.

Differentiation of HSCs to B lymphocytes involves progression through multipotent progenitor (MPP), lymphoid/myeloid-primed multipotent progenitor (LMPP) and common lymphoid progenitor (CLP) stages of development, accompanied by the sequential loss of megakaryocyte/erythroid (MEP) and myeloid potentials [1,2] (Figure 1). One powerful characteristic of the hematopoietic model system is the ability to isolate, based on surface phenotype, intermediates that represent developmental stage-specific progenitors. Using defined surface markers, studies over the last ten years have progressed from isolation of phenotypic subsets to isolation of single cells. A core transcriptional network that orchestrates B cell fate specification within sequential developmental contexts is established. The transcription factors Ikaros, PU.1 and E2A regulate lymphoid versus myeloid fate choice while EBF and Pax5 control B cell specification and commitment (Figure 2). Recent progress connects genetic and epigenetic mechanisms during specific developmental transitions between lymphoid progenitors and B cell precursors.

Figure 1.

Figure 1

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The development of B cells from HSCs involves progression to multipotent progenitor (MPP), lymphoid/myeloid-primed multipotent progenitor, common lymphoid progenitor, (CLP) and B cell progenitor intermediates prior to B cell commitment. In one current model of developmental progression, establishment of the B lineage fate is accompanied by sequential loss of megakaryocyte/erythrocyte (Mk/E) potential, myeloid potential and T/NK cell potentials.

Figure 2.

Figure 2

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Hallmark developmental transitions during HSC progression to the B cell fate. Indicated below each major phenotypic stage is the defining functional criteria, surface markers, key transcription factors, and associated target genes. Emphasis is placed on E2A to reflect recent scientific advances.

Here, we review current knowledge of transcription factors and their cis-targets critical for B lineage development. We integrate this transcriptional network within a broader context of recent genome-wide analyses that reveal the patterns of transcription factor binding at sequential developmental stages. Emerging from these studies is a clearer understanding of how the concerted binding of multiple transcription factors (trans-acting factors) at defined regulatory regions (cis-acting elements) regulates gene expression in a developmental stage-specific manner (phenotype). In other words, the gap between genotype and phenotype just got a little smaller.

Transcriptional regulation of B cell development

Progression of HSCs to the B lineage is marked by the loss of pluripotent self-renewal activity and activation of a B cell specific program. During this transition, characteristic low-level gene expression associated with multi-lineage priming gives way to increased expression of genes associated with the B cell fate [3,4]. Genes expressed during multi-lineage priming commonly exhibit bivalent chromatin marks in which both activating and inhibitory modifications to histones and DNA are present [5,6]. Within CD150+ LSK (lineage marker-negative, Sca-1+, c-kit+) stem cells, for example, ~4% of ebf and pax5 promoters bear coincident activating H3K4me3 and inhibitory H3K27me3 modifications [7**]. These bivalent histone marks are subsequently resolved concomitant with changes in the activity of transcription factors that upregulate lymphoid lineage genes and repress genes associated with alternative lineage fates.

The Ikaros transcription factor is a major regulator of HSC progression to the lymphoid lineages. Encoded by the Ikzf1 gene [8], Ikaros suppresses stem cell associated genes including the receptor tyrosine kinases tie1, tie2 and mpl, and induces lymphoid-specific genes including the Dntt nucleotide transferase [9**]. In a powerful experiment, the lymphoid induction potential of Ikaros was shown within single HSCs, the functional level at which lineage fate decisions are made [9**]. Ikaros activity is required in LMPPs and early B cell precursors where it regulates expression of the cytokine receptor flt3, the λ5 pre-B cell receptor chain [10] and rag1/2 genes. Ikaros also regulates immunoglobulin chromatin remodeling [11*]. One critical function of Ikaros is to antagonize the transcription factor PU.1 (purine box factor-1) to direct the lymphoid versus myeloid fate [12].

PU.1, an Ets family transcription factor, regulates gene targets important to both the lymphoid and myeloid lineages including receptors for the cytokines IL-7 and the granulocyte/macrophage (G/M) colony stimulating factors, respectively. PU.1 expression in MPPs restricts MEP fate [13] after which the coordinated interaction of PU.1 and the Ikaros-induced Gfi-1 (growth factor independent-1) transcription factor establishes B versus myeloid fate choice by stabilizing PU.1 levels [12]. PU.1 is maintained at low levels in B cells and at high levels in myeloid cells [14,15]. Engagement of PU.1 motifs within the Sfp1 promoter itself can promote autoamplification in a feed forward loop [16,17]. During lymphopoiesis however, Ikaros induces Gfi-1, which, in turn, blocks PU.1 autoamplification through physical displacement of PU.1 at the Sfp1 promoter [11*]. The biological effect is to reduce PU.1 expression to lymphoid-appropriate levels. In addition to limiting PU.1, Gfi-1 reinforces lymphoid progression by enhancing E2A activity through direct suppression of the inhibitor of DNA binding factor-2 (Id2), an E2A antagonist [18]. Finally, the ability of PU.1 to direct major downstream factors E2A, EBF, Oct-2 and NFκB to defined target genes in a lymphoid lineage-specific manner is one likely mechanism that favors lymphopoiesis versus myelopoiesis [19].

E2A, a basic helix-loop-helix transcription factor, is emerging as an important regulator of HSC self-renewal and of LMPP – but not HSC – lineage restriction. E2A deficient HSCs exhibit diminished self-renewal capability following adoptive transfer, hyperproliferation associated with loss of the p21 cell cycle inhibitor, and a failure to prime lymphoid lineage-associated genes [20**, 21, 22**, 23]. Loss of HSC activity is also observed in mice lacking either the E2A inhibitor Id1 [24,25] or the E2A interaction partner Stem Cell Leukemia (SCL) [26], suggesting an interacting network of E proteins at this stage. E2A is dispensable for the restriction of MEP and myeloid lineage potentials as assessed using single HSCs [20**,22**]. E2A may regulate later dynamics as erythroid and megakaryocyte intermediates are diminished [21]. MPPs and LMPPs are numerically reduced in the absence of E2A [20**,23], and downstream CLPs are virtually ablated [27]. In vivo administration of anti-oxidant restores MPPs but not the LMPP or CLP subsets in E2A deficient mice [22**], thereby defining new activities of E2A independent of lymphoid lineage differentiation.

E2A is an essential regulator of lymphoid specification and restriction of myeloid potential at the LMPP stage [20**]. Gain-of-function and loss-of-function studies show that the balance between E proteins and Id proteins regulates lymphoid versus myeloid fate decisions of LMPPs [28]. In addition to being reduced in number, remnant LMPPs from E2A-deficient mice fail to fully upregulate the Flt3 cytokine receptor [20**,22**] that is essential for development of Flt3hiVCAM-1−LMPPs [29]. E2A does not regulate flt3 transcription per se [20**]. Rather, Hoxa9 and PU.1 regulate flt3 expression, although cooperative interactions between E2A, hoxa9, and PU.1 in LMPPs remain to be established [30]. E2A-deficient LMPPs additionally lack V(D)J recombinase activity [27]. At the molecular level, E2A-deficient LMPPs have decreased expression of the lymphoid associated genes IL7Rα, rag1, Dntt, Igh-6, notch1 and ccr9 [20**], and fail to generate pro-B cells [31]. The E2A inhibitor, HEBalt, an alternative splice product of the HEB locus, emerges as a context-dependent regulator of cell fate choice. HEBalt inhibits B cell outgrowth to the benefit of myeloid cells, but in the presence of Notch ligands, favors T cells over myeloid cells [32].

In summary, three major roles for E2A are emerging. First, E2A appears to be required for efficient HSC self-renewal and persistence, and for lymphoid-lineage priming at this stage. Second, E2A promotes the production and/or maintenance of MPPs and LMPPs, and is required for myeloid restriction in LMPPs. Third, as detailed immediately below, E2A is required for promotion of B lineage progression through a cascade involving the EBF, Pax5 and Foxo1 regulatory factors.

EBF and Pax5 sequentially specify and commit lymphoid precursors to the B cell fate. Forced expression of EBF in MPPs activates the lineage appropriate genes pax5, lambda5, VpreB and Cd79b and represses genes associated with alternative fates including c/EBPα [33] (Figure 2). Binding of the interferon-regulatory factor IRF8 to the ebf promoter results in transcriptional activation of EBF while binding to the Sfp1 promoter represses PU.1 [34]. EBF, in turn, reinforces E2A activity by repressing the Id inhibitors of E2A [35]. Unlike E2A-deficient mice that lack CLPs, EBF-deficient mice have a numerically replete CLP compartment that lacks B lineage potential. At the single cell level, EBF KO CLPs fail to upregulate pax5, Pou2af1 and mb-1 transcripts [36]. Single cell tracing reveals that high levels of rag1 accompany loss of myeloid and NK potential [37**,38*]. Almost all raghi CLPs express EBF, with ~31% of these cells also expressing pax5 and retaining both B and T cell potential in vitro. T cell potential is lost concomitant with full transcriptional activity of EBF. Rag expression in CLPs correlates with Ly6D expression suggesting the value of this surface marker for distinguishing CLPs with global lymphoid potential versus CLPs that are B lineage biased [37**,39*]. Compound haploinsufficiency of EBF and RUNX1 diminishes B lineage specific gene expression resulting in a block in B cell progression [40].

Pax5 maintains the B cell fate [41**,42]. Conditional ablation of pax5 in CD19+ splenocytes leads to a loss of the B cell program and a striking conversion to the T cell fate. In support of these observations, Notch1 signaling in response to delta like ligand 4 leads to a dose dependent inhibition of Pax5 and the B lineage program in the thymus [43]. In humans, ablation of the E2A-EBF-Pax5 pathway is associated with Hodgkin lymphoma in which B cells exhibit a loss of identity uncannily reminiscent of pax5 conditional deletion [42].

Epigenetic regulation of the B cell development

Cell fate decisions are dictated by the activation state of core transcription factors and the access of these factors to select genomic regions. The epigenetic mechanisms that regulate B lineage determination are being revealed at the sequential levels of genome wide analysis (E2A, EBF), a promoter/enhancer complex (pax5), and B cell-specific genes (mb-1, Cd19).

Murre et al. detail the association of E2A binding to cis elements, epigenetic modification, coordinated occupancy by cooperating factors, and transcript abundance at sequential stages of B cell development [44**]. There are two thematic advances in this study. First, the data provide a molecular genetic snapshot of B cells at a developmental transition where they still possess a genotype identical with most other cells in the body yet have a distinct phenotype. Second, this study quantifies the positive effects on gene expression of cooperative transcription factor binding. E2A occupancy near transcription start sites is associated with activating H3K4 monomethylation marks at enhancers and H3K4 trimethylation marks at promoters (Figure 3). About half of the E2A-occupied sites in pre-pro B cells are also occupied in pro-B cells, but proB cells acquire ~10,000 new E2A-bound sites. E2A-occupied sites co-localize with binding motifs of factors essential to the B lineage fate including EBF and PU.1 [44**], the Foxo1 regulator of rag expression [45,46], the CTCF regulator of immunoglobulin locus contraction [47] and Bcl11a [48]. Co-occupancy by E2A and EBF correlates with H3K4 methylation status and an abundance of the hallmark B lineage transcripts VpreB, Cd19, foxo1, Pou2f1, Cd79a, and pax5. These data define a molecular genetic basis for the functional synergy observed between E2A and EBF [49]. Loci enriched for co-occupancy of E2A and Foxo1 include Dntt, the Erg transcription factor, and the pro-apoptotic gene bcl2l11 that encodes Bim. Mice doubly heterozygous for E2A and Foxo1 (e2A+/−foxo1+/−) have reduced B cell progenitors compared to either single heterozygote, thereby directly linking E2A, EBF and FOXO1 in a common pathway [44**]. In another example of lineage-specific cooperativity, the ability of PU.1 to bind to defined targets is influenced by the presence of cooperating factors as enforced expression of E2A increased PU.1 binding sites by 4-fold [19].

Figure 3.

Figure 3

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Epigenetic modification and transcription. Bivalent chromatin modifications mark B lineage-specific genes in HSCs. Such genes are considered poised but silent. E2A binding to cis elements is associated with activating H3K4 monomethylation at enhancers and H3K4 trimethylation at promoters. E2A activates EBF and Pax5, initiating a cascade of expression of B cell-specific genes. Transcription factor cooperation elevates the magnitude of gene expression.

Genome-wide analyses reveal EBF-activated and -repressed targets across defined stages of B cell development [50**]. EBF-activated targets (Cd79a, Gfra2 and pax5) gain the activation marks H3K9me and H3ac while EBF-repressed genes acquire repressive H3K27me3 marks. A subset of developmentally regulated genes (e.g., Cd40) exhibits the H3K4me2 poised mark at the pro B cell stage followed by the activating H3K4me3 and H3ac at the mature stage, suggesting that EBF primes chromatin prior to transcriptional activation. Enforced expression of EBF in a CD4+CD8+ T cell line or in NIH 3T3 cells reveals EBF capabilities for chromatin poising. In the T cell line, EBF binding and H3K4me2 modification is present at Cd79a, pax5 and Cd40 loci, with low-level transcription detectable from the Cd40 locus. In NIH3T3 cells, neither EBF binding nor activating histone marks are observed. Thus, EBF recognizes binding sites independent of transcriptional activation but not in closed chromatin [50**].

Busslinger et al. define the stepwise activation of the Pax5 locus. Pax5 expression is dictated by an enhancer in intron 5 of the pax5 locus and a promoter element [51**]. CpG motifs in the enhancer are demethylated during the HSC to MPP transition, and acquire the active chromatin mark H3K9ac+H3K27me3− by the pro B cell stage. The enhancer is positively activated by PU.1, IRF-4 and -8, and NFκB. By contrast, the promoter is demethylated even in HSCs. However, in the absence of E2A or EBF, the promoter fails to acquire the H3K9ac activating histone marks. These findings highlight a critical role for EBF in activation of the pax5 promoter.

Pax5 collaborates with other factors to remodel B lineage-specific targets mb-1 and Cd19. The mb-1 promoter undergoes demethylation during the HSC to pro B transition [52]. Demethylation requires the activity of both EBF and Pax5 as enforced expression of EBF in ebf−/−pax5−/− progenitors is not sufficient to mediate demethylation. Efficient transcription of mb-1 requires SWI/SNF activity and is suppressed by Mi-2NuRD chromatin remodeling complexes [52]. The CD19 locus undergoes stepwise activation of the enhancer and promoter [53]. E2A binding sites within the Cd19 enhancer are already demethylated as early as LSKs, suggesting that CD19 undergoes lineage priming well before the stage of lineage-specification. Progressive demethylation correlates with the successive binding of E2A, EBF and Pax5 in a developmental stage specific manner. CD19 transcriptional activation is achieved only after Pax5 binding to the promoter, an interaction accompanied by H3K4 trimethylation. Both studies detail the mechanistic importance of transcription factor cooperativity in lineage priming and the stable expression of B cell-specific genes, echoing a major theme of this Review.

Summary and Perspective

The ability to link coordinated changes in gene expression to developmental potential is essential for a clear vision of the mechanistic factors that drive lineage fate choice. Research progress over the last ten years has defined a core network of transcription factors that activate the B cell fate and repress alternative fates. We now add to this foundation an ability to discern the epigenomic marks that influence gene expression. The studies highlighted here describe epigenetic changes associated with HSC progression to the B cell fate at the hierarchical levels of genome-wide analysis, B lineage-specific transcription factor activation, and B cell locus-specific gene expression. On the horizon is the opportunity to evaluate chromatin poising and gene expression within single cells, the biological level at which lineage fate decisions are made. The most significant advances are likely to come from progenitors poised at critical developmental transitions. Observations that lineage priming within HSCs is associated with bivalent histone states raises major questions. On a per cell basis, do individual HSCs bearing identical bivalent marks have comparable potential for lineage progression? Or, are some HSCs refractory to lineage progression due to other limiting co-modifications? HSCs with heterogeneous development potential appear to co-exist in the bone marrow, with some stem cells expressing lymphoid bias, others expressing myeloid bias, and still others possessing a latent potential that manifests in robust repopulation activity only after secondary transplantation [54]. Future studies will reveal how chromatin status and gene expression patterns of individual HSCs – or cells representing later development stages – track with lineage reconstitution potential and progression to the B cell fate.

Acknowledgments

Supported by NIH R01 AI079047 with seed perspective developed under an NSF/Alfred P. Sloan Foundation Fellowship in Molecular Evolution (LB). We deeply appreciate direct input from K Murre, M Sigvardsson and K Medina.

Footnotes

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