To maintain homeostasis of hematopoietic cells, HSCs must undergo divisions that ensure that one or both daughter cells remain in an undifferentiated state. In the mesoderm, the transcription factor SCL dimerizes with the E proteins to commence a hematopoietic program. In the developing embryo, HSCs are primarily localized in the fetal liver whereas in the adult they are localized in the bone marrow. Recent studies have begun to identify components that promote HSC survival. For example, forced expression of the anti-apoptoptic factor Bcl2 increases HSC self-renewal and transplantation efficiency. In contrast, deletion of the anti-apoptosis factor Mcl1 leads to the complete failure of hematopoietic development, indicating its necessity for HSC's survival.
The Hox family of transcription factors also plays a critical role in hematopoietic development and HSC self-renewal. The Hox genes were identified in Drosophila as regulators of body patterning pathways, and have since been shown to be required for mammalian hematopoiesis. The Hox proteins interact with the co-factor Meis1 to direct normal hematopoiesis. Although multiple Hox genes are expressed in HSCs, Hoxa9 is expressed at very high levels. Hoxa9−/− mice exhibit multiple hematopoietic defects including the inability of Hoxa9−/− HSCs to properly reconstitute. In contrast, Hoxb4 deficient mice lack a dramatic hematopoietic phenotype even though overexpression of the Hoxb4 gene allows for increased self-renewal and expansion of HSCs in vitro.
The zinc-finger repressor protein Gfi1 (growth factor independence 1) is also required for HSC self-renewal. Initially, roles for Gfi1 were identified in both T-cell development and T-cell lymphomas. A role for Gfi1 in HSCs was confirmed by assays showing that HSCs from mice deficient for Gfi1 have increased rates of self-renewal and cannot effectively reconstitute animals in transplantation assays. Gfi1 activates expression of the cell cycle inhibitor p21CIP1/WAF1, which is also required for normal hematopoiesis. However, p21 and Gfi1 knockouts have differing hematopoietic defects, indicating that although Gfi1 may act in part through regulation of p21, it must have other actions of HSC regulation as well. Interestingly, recent work has connected the stress response to HSC self renewal by linking p53 activity and Gfi1 expression.
The polycomb group proteins (PcGs) are critical for efficient HSC self-renewal. Specifically, Bmi1 is required for HSC homeostasis, as animals deficient for Bmi1 show a severely reduced self-renewal capacity and an increased rate of differentiation. Correspondingly, overexpression of Bmi1 in HSCs leads to increased self-renewal in vitro and increased repopulating ability in vivo .
Signaling pathways that are active in controlling HSC self-renewal include the JAK–STAT pathway and Wnt signaling. Overexpression of the JAK activated transcription factor STAT5 leads to increased self-renewal and expansion of hematopoietic progenitors ex vivo, and HSCs from STAT5 knockout mice have a decreased ability to reconstitute irradiated recipients. Addition of Wnt proteins to culture media leads to increased self-renewal of HSCs in vitro, and HSCs expressing a constitutively active form of β-catenin exhibit improved self-renewal and reconstitution ability in vivo .
HSCs express not only transcription factors that maintain their self-renewal capacity, but also transcription factors closely associated with the development of multiple lineages. This lineage priming is suggested to reflect the pluripotent capability of these cells. It has been shown that HSCs express many myeloid required transcription factors, including MPO, CEBPα, and MCSFR. Erythroid and megakaryoctye required factors such as Gata1, EpoR and MPL are also expressed in the most primitive of bone marrow hematopoietic precursors. Upon differentiation into MPPs and LMPPs, the mRNA levels of the erythroid specific genes decrease, while the levels of the myeloid transcription factors remain stable. Appreciable expression of lymphoid specific genes such as preTα and Pax5 is not seen until the CLP stage. This suggests that the “default” lineages for the hematopoietic system lean toward the innate immune system, and that lineage commitment occurs upon both the upregulation and activation of a lineage specific gene network with the concomitant downregulation or suppression of transcription of genes associated with alternative cell lineages.
During the past two decades substantial insight has been obtained about the participants that promote the development of the B cell lineage and their concerted activities. The transcription factor Ikaros was among the first transcriptional regulators shown to play an important role in orchestrating lymphoid development. Ikaros is a Kruppel-like zinc finger protein that functions as a dimer or multimer to recruit either co-repressor or co-activator complexes such as the SWI/SNF nucleosome remodeling complex. Ikaros−/− mice have defects in HSC self-renewal and lymphoid development beyond the LMPP cell stage. Specifically, Ikaros−/− LMPPs fail to upregulate Flt3. Thus, CLPs are absent in Ikaros ablated animals, leading to a block in B cell development and an increased potential for NK cell development. Ikaros has also recently been shown to be necessary to allow V-DJ recombination in pro-B cells. Specifically, infection of Ikaros−/− LSK progenitors with a retrovirus expressing EBF allows progression to the CD19+ B-cell stage, but is not sufficient to permit V-DJ recombination to occur. In fact, CD19+ EBF expressing Ikaros−/− cells can be diverted to the myeloid lineage upon appropriate cytokine stimulation. Thus, Ikaros seems to play multiple roles in the development of B cells including: the development of the CLP through regulation of Flt3 and, the control of V-DJ rearrangement through regulation of the Rag recombinase genes and accessibility at the IgH locus.
It has been suggested that Ikaros regulates expression of Flt3 and IL7R in concert with PU.1. PU.1 is an ETS family transcription factor that is required for development of both myeloid and lymphoid lineages. PU.1 is expressed in HSCs, LMPPs, CLPs, myeloid and B cell progenitors, and in developing T, NK and dendritic cells. Mice deficient for PU.1 die soon after birth and lack myeloid and lymphoid development, but show normal erythroid development. PU.1 has specific roles in the bifurcation of the myeloid and B cell lineages. High levels of PU.1 enforce myeloid development, while low levels promote B-cell differentiation. Control of this PU.1 gradient for B cell commitment appears to be through direct repression of PU.1 by Gfi1. Gfi1−/− mice have reduced B cell potential, as the loss of the repression of PU.1 diverts cells to the myeloid lineage. Interestingly, Gfi1 transcription is regulated by Ikaros, suggesting a transcriptional loop to support B-cell development and repress the myeloid fate choice.
The E2A proteins are also implicated in the expression of the IL7Rα gene. The E2A gene encodes two splice variants, E12 and E47, and is a member of the basic helix–loop–helix E protein family of transcription factors. In mice lacking the E2A gene, there is a complete block in B-cell development at the BLP stage before IgH D-J rearrangement has occurred. Recently, it has been shown that E2A plays a distinct role in the HSC and LMPP compartments, as there is a significant decrease in the numbers of HSCs and LMPPs in E2A heterozygous and knockout animals. E2A also has specific roles in rearrangement of the heavy and light chain genes at the pro-B and pre-B stages of development. Additionally, E2A directly binds and upregulates the transcription factors EBF1, Foxo1, Pax5, IRF4, IRF8 as well as other target B-cell genes, to activate a B-lineage program of gene activity. Although E2A has a distinct role in positively promoting B-cell development, it also acts to suppress the expression of genes associated with alternative cell lineages. E2A is inhibited by the Id proteins, which heterodimerize with E proteins at the helix–loop–helix domain to inhibit their ability to bind DNA.
The EBF transcription factor contains a HLH motif used for dimerization and an N-terminus zinc coordination motif that mediates DNA binding. Mice deficient for EBF have an almost identical block in B-cell development as the E2A−/− mice. EBF−/− mice also fail to express B-cell specific genes, and do not have IgH rearrangement. EBF is a transcriptional target of E2A and is sufficient to rescue B-cell development when expressed in E2A−/− progenitors. Mice that are heterozygous for both E2A and EBF have more severe B-cell phenotypes than the single heterozygotes, suggesting a synergistic activity for E2A and EBF. Recently, a synergistic relationship between Runx1 and EBF has also been shown to be critical to permit B-cell development, as the double heterozygous animals show defects in differentiation of CD25+ pre-B cells and stunted activation of B cell specific genes.
EBF is regulated by both a distal α promoter and a proximal β promoter. The α promoter is active early in B-cell development through both direct E2A binding and indirect activity of STAT5, which is activated through the IL-7R. Additionally, there is an EBF binding site at the α promoter, suggesting that EBF is also regulated through an autoregulatory loop. The β promoter is active in more mature B cell populations, and is activated by binding of Pax5, Ets1 and PU.1.
The transcription factor Pax5 is regulated by E2A, EBF, IRF4, IRF8 as well as PU.1. It plays an essential role in B-lineage commitment. Pax5 is only expressed in the B-cell lineage, where it is turned on in pro-B cells and remains on until the plasma cell stage. B cell development is arrested in Pax5−/− mice at the pro-B cell stage of development. While EBF is required to induce the expression of Pax5, Pax5 induces the expression of EBF in a feedback loop. It has recently become evident that both EBF and Pax5 act to promote commitment to the B cell lineage.
Pax5 can act both as a transcriptional activator and a repressor. Consistent with these activities, Pax5 has been shown to activate a B-cell specific program of gene expression while directly suppressing alternate lineage specific genes. Evidence suggests that Pax5 represses myeloid genes through antagonistic associations with the myeloid factor CEBPα. In the absence of Pax5, pro-B cells maintain an unusual plasticity in that they can develop into myeloid cells and T cells.
Recently, additional transcription factors that play essential roles in B-cell development have been identified. Mice with B cell specific deletions of Foxo1 are arrested at the pro-B cell stage. Specifically, Foxo1 was shown to be required for expression of IL-7R and Rag. Furthermore, mice that are heterozygous for both E2A and Foxo1 have an almost complete absence of B cells. Global patterns of Foxo1 occupancy in pro-B cells have indicated that E2A, EBF and Foxo1 act coordinately at enhancer elements to activate a B-lineage program of gene expression. The Kruppel-related zinc finger protein Bcl 11a is also required for B-lymphopoiesis. Mice deficient for Bcl 11a have a block before pro-B cell commitment. Genetic analysis revealed that Bcl 11a−/− fetal liver progenitors do not express IL7R, EBF, Pax5 or CD19, suggesting that Bcl 11a may act in concert with E2A to initiate the B-cell fate. Additionally, the genome-wide studies described above have demonstrated that the E2A proteins bind to putative enhancer elements present in the Bcl1A locus.
During early hematopoiesis, developmental pathways with multiple branch points from which progenitor cells give rise to multiple hematopoietic lineages have been specified. Transcription factors critical to these pathways have been identified to characterize and modulate both the developmental progression and expansion and survival of early progenitors. However, it remains largely unknown as to how these transcription factors are linked to promote lineage- and stage-specific phenotypes. Recent studies using ChIP-Seq approaches, along with new computational analyses like HOMER (Hypergeometric Optimization of Motif EnRichment), have provided mechanistic insight into how myeloid- and B-lineage specification is established at a global scale.
As discussed above, the activities of PU.1 are critical to promote both myeloid- and B-lineage specification. Recent genome-wide studies demonstrate how PU.1 acts at a global scale to modulate lineage choice. In myeloid cells, PU.1 binding was primarily associated with consensus binding sites for AP-1 and C/EBP. In contrast, promoter-distal PU.1 occupancy in B-lineage cells was predominantly enriched at genomic regions containing promoter-distal E2A, PU.1:IRF, EBF, NFκB, and OCT consensus binding sites. In both myeloid and B cells, PU.1 genome-wide occupancy was predominantly detected at promoter-distal sites, and was tightly correlated with myeloid- and B-lineage specific programs of gene expression, respectively.
Similar studies have been performed studying E2A occupancy at the genome-wide scale. In pre-pro-B cells, enhancer elements associated with E2A occupancy were significantly enriched for RUNX and ETS consensus binding sites. In contrast, in pro-B cells, E2A bound sites were primarily associated with ETS, EBF, RUNX, and Foxo1 binding sites. Furthermore, Foxo1 and EBF ChIP-Seq experiments show that coordinate E2A, EBF and Foxo1 DNA binding sites were closely correlated with a B-lineage program of gene expression. Similar ChIP-Seq studies that analyzed only EBF1 binding showed a significant enrichment for the ETS, E2A, EBF1 and Pax5 binding sites. In the latter study, it was observed that EBF1 occupancy showed a tendency to associate with components involved in pre-BCR mediated signaling.
These studies have generated a global network of transcription factors proposed to orchestrate the B cell fate. Briefly, analysis suggests that in progenitor cells, the E2A proteins directly induce the expression of EBF1, IRF4, IRF8 and Foxo1. Additionally, E2A may regulate the expression of Pax5 through multiple circuitries. Previous studies have indicated that IRF4, IRF8 and PU.1 act to induce the expression of Pax5. The E2A proteins directly interact with putative enhancer localized within the Pax5 locus, but they also modulate Pax5 transcription by the induction of Foxo1, Ebf1, IRF4 and IRF8 expression.
The construction of a network that underpins specification of the B cell lineage also revealed new connections. E2A was found to directly bind enhancers present in the Bcl 11A and CTCF loci, linking E2A, Bcl 11A, and potentially CTCF into a common pathway. During developmental progression from the pre-pro-B to the pro-B cell stage, the IgH locus undergoes large-scale conformational changes, and it is conceivable that the activation of CTCF expression contributes to IgH locus contraction. It was also demonstrated that E2A and Foxo1 directly bind to potential regulatory elements present in the LEF1 locus. The LEF proteins have been shown to act downstream of the Wnt signaling pathway and modulate B cell expansion, thus linking E2A, Foxo1 and Wnt signaling into a common pathway. The suppression of genes associated with alternate cell lineages was also found to be associated with differing combinations of E2A, EBF and/or Foxo1 occupancy. Among these genes are regulators that play critical roles in early hematopoeisis including CEBPα, Notch1, RUNX2, RUNX3 and GATA3. These data raise the question as to how E2A, EBF and Foxo1 act to either activate or inhibit the expression of genes. It may be suggested that this is dependent on the context of the regulatory elements to which they bind. It is conceivable that the spatial arrangement of various cis-regulatory elements determines the outcome of combinatorial interactions to either activate or suppress downstream target gene expression.
Genome-wide DNA binding studies of transcriptional regulators and global patterns of gene expression have provided new insights into how hematopoietic development is orchestrated. New players were identified and new connections have been revealed. These approaches are only a first step. The global DNA binding patterns of other transcriptional regulators that play critical roles in orchestrating lineage choice, including Pax5, PU.1, IRF4, IRF8, Bcl11A and LEF need to be incorporated. Further studies should be combined with gene-ablation strategies performed at specific developmental stages. Better strategies to assign regulatory elements to specific loci need to be developed. This will require further insights into chromosome structure and how this relates to long-range genomic interactions. We are just at the beginning of gaining insight into the global mechanisms that underpin developmental progression and lineage choice.