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Gene targeting reveals a hierarchy of transcription factors regulating specification of lymphoid cell fates Harinder Singh

Specification of B- and T-lymphoid cell fates appears to involve the expression of a shared set of genes encoding recombination proteins and of genes encoding lineage-specific components of antigen receptors. Recent studies using gene targeting have identified transcription factors that are required for the proper specification of lymphoid cell fates. On the basis of these data, a regulatory gene hierarchy which orchestrates the development of lymphoid progenitors from hematopoietic stem cells can be proposed.

Address Howard Hughes Medical Institute, The University of Chicago, 5841 South Maryland Avenue, MC 1028, Room N-112, Chicago, IL 60637, USA; e-mail hsingh@midway.uchicago.edu

Current Opinion in Immunology 1996, 8:160-165

© Current Biology Ltd ISSN 0952-7915

Abbreviations EBF early B-cell factor mlg membrane-bound Ig pre precursor pro progenitor SFFV spleen focus-forming virus TCR T-cell receptor

I n t r o d u c t i o n The development of B and T lymphocytes from their respective progenitors is a highly ordered and coordinated process. Each pathway of development is characterized by regulated patterns of gene expression as well as DNA rearrangements involving immunoglobulins or TCR loci [1,2]. Developmental intermediates can be isolated and analyzed on the basis of staged expression of cell-surface markers [3]. B-lineage progenitor (pro-B) cells uniquely express a set of four genes: mb-1, B29, L5 and Vpre-B. The mb-1 and B29 genes encode the Ig-et and Ig-13 components, respectively; they are required for cell-surface transport and signal transduction by membrane-bound Ig (mlg). The L5 and Vpre-B genes encode surrogate light chains. They associate with the I1 polypeptide (product of Ig heavy-chain gene rearrangement) in precursor (pre) B cells to generate the pre-B receptor complex. This complex is needed for efficient development of B lymphocytes expressing an antigen receptor.

As is the case for their B lymphoid counterparts, T-lineage progenitor (pro-T) cells uniquely express genes encoding auxiliary components of the TCR. These include subunits of the CD3 complex that are required for cell-surface assembly and signalling by the TCR. In addition, T-lineage progenitors that are specified to give rise to ~13

T cells express the co-receptors CD4 and CD8. Both pro-B and pro-T cells express genes encoding the recombination apparatus, Rag-l, Rag-2 and TdT, which are required for antigen receptor gene rearrangement and diversification. Expression of these genes distinguishes lymphoid progen- itors from myeloid, erythroid and megakaryoid progenitors in the hematopoietic system. Thus, the specification of lymphoid cell fates appears to involve both the expression of a shared recombination apparatus and the expression of components of antigen receptors that are lineage specific.

Gene targeting in mice has been used to assess the roles of antigen receptor components, cytokines and their receptors and signalling molecules in lymphocyte development and function [4]. More recently, the same approach has been applied to a set of transcription factors that were implicated in regulating lymphocyte-specific gene expression. This review focuses on genes encoding transcription factors, in which loss-of-function mutations result in either elimination of both B and T lymphoid progenitors or the selective loss of one lineage. These genes encode the transcription factors PU.1, Ikaros, E2A, EBF and BSAP/Pax-5. The mutant phenotypes suggest that these transcription factors act in a hierarchical manner. On the basis of the results, a regulatory gene hierarchy orchestrating the development of lymphoid progenitors from hematopoietic stem cells is proposed.

PU.1 The transcription factor PU.1 is an ets domain protein that is specifically expressed in the hematopoietic system [5,6]. PU.1 is the product of the Spi-1 proto-oncogene [7]. The Spi-1 locus is the site of integration of the spleen focus-forming provirus (SFFV) in 95% of murine erythroleukemias induced by Friend virus complexes [8]. PU.1 is expressed at high levels, particularly in the mono- cytic and B-lymphoid lineages. Numerous presumptive target genes have been identified in each of these two lineages. In the B lineage, PU.1 is implicated in regulating transcription of the Ig heavy (I-t) [9] and light chain genes (K and ~.) [10,11], the mb-1 gene [12,13] and the J-chain gene [14].

A mutation in the PU.1 gene was engineered by delet- ing the exon sequence encoding the ets DNA-binding domain [15"']. This mutation causes late embryonic lethality. Analysis of fetal hematopoiesis in PU.1 mutant embryos revealed an invariant multi-lineage defect in the generation of B and T lymphocytic, monocytic and granulocytic progenitors. For each lineage, analysis with multiple stage-specific markers failed to reveal any differentiating intermediates. As erythroid progenitors

Transcription factors regulating specification of lymphoid call fates Singh 161

and megakaryocytes are generated in normal numbers in mutant embryos, the multi-lineage defect is not simply a reflection of a critical function for PU.1 in the hematopoietic stem cell. Thus, PU.1 is a unique regulator of hematopoiesis, being specifically required for the development of the lymphoid and myeloid lineages. PU.1 is necessary for the specification of lymphoid progenitors. Its mutation results in elimination of B220 ÷, CD43 ÷ pro-B cells and Thy-1 +, CD2÷/CD4 ÷, CD8 ÷ pro-T cells. In the B lineage, the mutation also results in loss of progenitors that express the genes mb-1, L5 and VpreB as well as Rag-1 and Rag-2. Furthermore, neither transcription nor rearrangement of immunoglobulin genes is detectable in mutant embryos.

The simplest interpretation of the multi-lineage defect in PU.1 mutant embryos is that the lymphoid and myeloid lineages share a common progenitor. According to this model, PU.1 is required for either the generation of a multipotential lymphoid-myeloid progenitor or its subsequent differentiation (see Fig. 1). In vitro clonogenic assays have demonstrated the existence of multipotential progenitors in the fetal liver (AA4.1 ÷, lin-) with both B-lymphoid and myeloid potential [16]. Such a progenitor population is significantly reduced in number in PU.1 mutant embryos (R Fisher, H Singh, unpublished data). Moreover, in contrast with their wild-type counterparts, PU.1 mutant progenitors (AA4.1÷, lin-) fail to proliferate or differentiate in vitro in response to signalling by interleukin-7 and stromal cell contact. As PU.1 appears to be expressed specifically in cells of the hematopoietic system and not in the interacting stromal cells, PU.1

Figure 1

is presumed to function in a cell-autonomous manner. Chimeric mice generated with PU.1 mutant embryonic stem cells or hematopoietic progenitors have established that PU.1 functions in a cell-intrinsic manner to specify lymphoid and myeloid cell fates (E Scott, H Singh, unpublished data). PU.1 may regulate the differentiation of lymphoid and myeloid progenitors by controlling the expression of both regulatory as well as structural genes. Some of the transcription factors discussed below may represent direct targets for regulation by PU.1. In addition, by regulating the expression of structural genes such as mb-1 and the Ig heavy chain locus in the B lineage, PU.1 may directly participate in lymphocyte specification.

Ikaros The Ikaros gene encodes several related zinc-finger- containing transcription factors (Ik-1 to Ik-5) that are generated by alternative splicing [17"']. During em- bryogenesis, Ikaros transcripts are expressed specifically in hematopoietic tissues [18]. The gene is expressed in both B- and T-lineage cells. High-affinity binding sites for Ikaros proteins have been characterized in regulatory regions of B- and T-lineage genes required for specification of lymphoid fates. These genes include those encoding components of the CD3-TCR complex, Rag-l, TdT, Ig heavy-chain locus and mb-1. On the basis of an initial finding that an Ikaros isoform could bind and activate a CD36 gene enhancer, the Ikaros gene was suggested to play a key role in T-cell specification.

A mutation in the Ikaros gene was engineered that deleted the exon sequence encoding three zinc-finger

Regulatory hierarchy of transcription factors that orchestrate the development of the lymphoid system. Single ovals denote experimentally defined progenitor cells; double ovals denote proposed multipotent progenitor cells. B, B lymphocyte potential; E, erythrocyte potential; G, granulocyte potential; HSC, hematopoietic stem cell; L, lymphocyte potential; Meg, megakaryocyte potential; M, monocyte potential; T, T lymphocyte potential, c-myb, PU.1 and Ikaros are proposed to be required either for the generation of the depicted multipotent progenitor or its subsequent differentiation.

Megakaryocytes ~ ~ " z . . . ~ ~

Erythrocytes ~ ~ O I k a r o s

Monocytes B E s B A F p ~ ~

B cells T cells © 1996 Current Opinion in Immunology

162 Lymphocyte development

domains required for high-affinity DNA recognition by the isoforms Ik-1, -2, -3 and -4 [17"']. The majority of Ikaros mutant mice died within one to three weeks of age. Analysis of hematopoietic tissue revealed that the mutation causes a selective loss of B and T lymphocytes and their progenitors. Erythroid, megakaryoid and myeloid cells were generated in Ikaros-mutant animals. Indeed, the frequency of erythroid and myeloid cells in the bone marrow and spleen was higher in mutant animals compared with wild-type litter mates. Thus, as is the case for PU.1, mutation of the Ikaros gene results in a profound block to lymphocyte development. Mutant thymic tissue is hypocellular and lacks any definable T-lineage progenitors expressing the cell-surface markers Thy-1, CD3, CD4 or CD8. Ikaros mutant bone marrow lacks the B220 ÷, CD43 ÷ pro-B population.

On the basis of its unique mutant phenotype, the Ikaros gene has been suggested to control the development of a bipotential, lymphoid-restricted progenitor from the hematopoietic stem cell [17"°]. The relationship between the transcription factors PU.1 and Ikaros, both of which regulate the specification of lymphoid progenitors, remains to be elucidated. As Ikaros regulates the development of a subset of hematopoietic lineages that require PU.1, the Ikaros gene is positioned downstream of PU.1 in the regulatory hierarchy (see Fig. 1). PU.1 could regulate iymphopoiesis by controlling the expression of the Ikaros gene. This possibility appears unlikely as wild-type levels of Ikaros transcripts are detected in PU.1 mutant embryos (E Scott, A Dey, H Singh, unpublished data). It seems, therefore, that PU.1 and Ikaros may be required in a combinatorial manner for specifying lymphoid cell fates.

E2A The E2A gene encodes two isoforms, El2 and E47, that are prototypical members of the helix-loop-helix family of transcription factors [19]. Members of this family of transcription factors bind to their regulatory DNA sites as homo- or heterodimers. The E2A gene and the related genes E2-2 and HEB are expressed ubiquitously whereas other members of this gene family, including the myogenic regulators, are expressed in a tissue-specific manner [20]. Importantly, even though E2A proteins are detectable in a variety of cell types, they appear to specifically form homodimers in B-lineage cells [21,22]. Two experimental approaches have implicated products of the E2A gene in regulating B-cell development. E2A proteins were initially identified as transcription factors that bound to functionally important regulatory sites in immunoglobulin heavy- and light-chain gene enhancers. Secondly, overex- pression of E47 in a pre-T-cell line was shown to induce transcription of the Rag-1 and unrearranged Ig heavy-chain genes as well as rearrangement of D H and JH segments.

Two different mutant alleles of the E2A gene have been engineered using gene targeting in embryonic stem cells [23"°,24"']. One mutation selectively disrupts the

exon encoding the DNA-binding domain of El2 whereas the other mutation deletes exons encoding DNA-binding domains of both the El2 and E47 products. In spite of these differences in the targeted alleles, the mutant mice have very similar phenotypes. This is explained by the fact that disruption of the El2 exon inhibits expression of E47 transcripts. Mutation of the E2A gene results in postnatal lethality, with a majority of the mutant mice dying within a week of birth. Detailed analysis of hematopoiesis shows that homozygous mutant mice specifically lack B-lineage cells. The block to B-cell development caused by the E2A mutation virtually eliminates the B220 +, CD43 + progenitor population. This phenotype is shared by the PU.1 and Ikaros mutations. Molecular analysis of E2A mutant fetal liver samples reveals a block to DH JH rearrangement in the Ig heavy-chain locus as well as a dramatic reduction in I~ Rag-l, mb-1, CD19 and k5 transcripts. Thus the E2A gene is required for specification of the B lineage. The selective requirement of E2A for B-cell development places it downstream of Ikaros and PU.1 in the regulatory gene hierarchy (see Fig. 1).

E2A proteins may regulate B-cell development by di- rectly controlling the expression of immunoglobulin and Rag genes as well as by controlling the expression of downstream regulatory proteins such as BSAP/Pax-5 (see below). The requirements of E2A for efficient Rag-1 gene expression in B-lineage cells, coupled with its dispensability for T-cell development, strongly suggests that Rag gene expression in B- and T-lineage progenitors is independently regulated. This does not necessarily contradict the existence of a shared lymphoid progenitor in the hematopoietic system in which Rag as well as Tar/" gene expression has been activated at low levels (see Fig. 1). In this model, further specification of the common lymphoid progenitor into the B or T lineage would involve upregulation of Rag gene expression, which is required for rearrangement, via lineage-specific pathways. This possibility is supported by evidence of low- and high-level states of Rag gene expression (the latter correlating with rearrangement) in developing lymphoid cells [25].

Early B-cell factor Early B-cell factor (EBF) is a tissue-specific transcription factor whose DNA-binding domain represents a novel type of zinc coordination motif [26]. In the B lineage, EBF activity is detected at all developmental stages except in terminally differentiated plasma cells [13,27]. It was suggested that EBF is a key regulator of mb-1 gene expression on the basis of binding to a functionally important site in the mb-1 gene promoter.

The EBF gene has been mutated by deleting a genomic sequence that encodes a functionally important portion of the DNA-binding domain [28°°]. Homozygous mutant mice were viable although -30% of them died by four weeks of age. The mutation resulted in a selective elimination of B-lineage cells. Interestingly, pro-B cells

Transcription factors regulating specification of lymphoid cell fates Singh 163

expressing B220 and CD43 are present in EBF mutant bone marrow but all further developmental intermediates are eliminated. Molecular analysis of the B220 ÷ population from EBF mutant bone marrow showed that these cells do not express transcripts from the rob-l, B29, VpreB, L5, Rag-1 and Rag-2 genes; however, normal expression of the germline Ix locus and somewhat reduced expression of the T d T gene was detected. Thus, EBF is required for proper development of B-lineage progenitors. EBF appears to act later in the B-lineage developmental pathway than E2A, Ikaros and PU.1, as B220 ÷, CD43 ÷ intermediates transcribing the germline ~t locus are generated in its absence.

BSAP/Pax -5 BSAP was initially described as a B-cell-specific activator protein with an expression pattern similar to EBF [29]. It is expressed in pro-B, pre-B and B cells but not in terminally differentiated plasma cells. Purification of the protein and cDNA cloning showed BSAP to be a product of the Pax-5 gene, which is expressed in the developing central nervous system as well as in B-lymphoid tissues [30]. BSAP binding sites have been characterized in the CD19 [31], VpreB [32] and Ig heavy-chain genes [33].

Targeting of the Pax-5 gene shows it to be required for proper B-cell development [34"']. Homozygous mutant mice die within three weeks of birth and also exhibit defects in mid-brain development. As is the case for the EBF mutation, development of B-lineage cells is arrested in Pax mutants at the stage of B220 ÷, CD43 ÷ progenitors. Molecular analysis of these intermediates has not been reported. Pax-5, however, appears to act downstream of EBF, as its expression is not detected in EBF mutant B-lineage progenitors [28"'].

Regulatory gene hierarchy specifying lymphoid cell fates Interpretation of the PU.1 and Ikaros mutant phenotypes and the c-Myb phenotype suggest a hierarchical scheme for the development of lymphocytes which invokes ordered and sequential generation of lineages in the hematopoietic system (Fig. 1). This scheme is a variant of a model originally proposed to account for the differ- entiated states of various leukemic cell lines [35]. The new genetic data are in agreement with the predictions of the original model. The sequential model invokes progressive restriction of the developmental potential of the stem cell, mediated by a hierarchy of transcriptional regulators. Furthermore, it differs from the classical model [36] by positing that the lymphoid and myeioid lineages are generated from a common multipotential progenitor.

Mutation of the c-myb locus blocks the development of erythroid and myeloid lineages at the earliest identifiable stages [37]. Lymphoid progenitors also appear to be eliminated by the mutation (ML Mucenski, personal com-

munication). Importantly, megakaryocytes are generated at wild-type levels in the fetal liver of mutant embryos. PU.1, like c-myb, is necessary for the development of lymphoid and myeloid progenitors; however, PU.1 mutant embryos exhibit wild-type levels of megakaryocytes as well as erythroid progenitors. Finally, Ikaros is exclusively required for the development of lymphoid progenitors.

Each of these studies has used both morphological as well as molecular markers to document the elimination or severe reduction of the earliest defined progenitors of the affected lineages. The multi-lineage progenitor defects induced by these three mutations are most simply interpreted as being attributable to a failure either to generate an appropriate multipotential progenitor or to sustain its differentiation. Although it remains possible that each of these transcription factors is separately required in committed progenitors of each lineage, this is considered unlikely as it would necessitate their function at equivalent steps in differing developmental pathways.

The existence of a muhipotential lymphoid-myeloid progenitor is supported by data from clonogenic assays in vitro which have revealed progenitors with both myeloid and B-lymphoid potential in the murine fetal liver [16]. Furthermore, bone-marrow-derived clones representing such an intermediate in the hematopoietic system have also been described [38]. A common origin for the myeloid and lymphoid lineages is also particularly attractive from an evolutionary standpoint, as the former lineages represent a more primitive immune system lacking antigen specificity. It should be noted that this scheme is not necessarily contradicted by earlier in vivo (CFU-S) and in vitro (CFU-GEMM) hematopoietic colony-forming assays. These assays reveal multipotential progenitors that can give rise to granulocytes, erythrocytes, megakaryocytes and monocytes. It remains possible that these progenitors are pluripotent and have lymphoid potential that is not revealed under the assay conditions. B-lineage progenitors are critically dependent on contact with stromal cells for their development, and likewise T-lineage progenitors require the thymic micro environment for differentiation.

Conclusion The genes described above represent a key set of regulators required for specification of lymphoid cell fates. Mutations in these genes affect the expression of the recombination apparatus as well as expression of antigen receptor components. In this regard, these mutations differ from those in Rag-1 and Rag-2 [39,40], and define earlier acting functions in lymphoid developmental pathways. B- and T-lineage progenitors are generated in the absence of Rag-1 or Rag-2 but they fail to differentiate further because of their inability to recombine Ig or TCR loci. Whereas PU.1 and Ikaros are required for the development of both pro-B and pro-T cells, the E2A, EBF and BSAP gene products are uniquely needed for B-cell specification. The existence of a set of transcription factors

164 Lymphocyte development

that are uniquely required for T-cell specification seems quite likely, although none has been described so far.

Although the gene-targeting approach has provided a very powerful means of defining a set of transcription factors required for the generation of B and T lymphoid progenitors, this approach has several limitations. It can be used to establish the requirement for a given regulator in development but not to demonstrate its sufficiency. The latter criterion is satisfied by pursuing ectopic expression experiments involving the regulatory molecule. Furthermore, no phenotype can be revealed by gene targeting in the presence of a redundant set of regulators. This problem can be circumvented in several ways, including the use of dominant negative mutants. Finally, even though multi-lineage developmental phe- notypes suggest the existence of common progenitors, cell marking experiments are necessary to validate lineage relationships.

In the near future, one can expect the lymphoid regulatory gene hierarchy to be elucidated further with the genetic analysis of additional transcription factors. Two key areas of investigation will involve examination of the interactions among regulators in the hierarchy and of the precise molecular basis of the lymphoid developmental defects induced by mutations of transcription factors.

Acknowledgements l thank Ed Scott, Celeste Simon and John Anastasi for stimulating discussions and Gall Hubbard for preparation of the manuscript.

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