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Concise Review |
Institute for Human Therapy, University of Pennsylvania, Philadelphia, Pennsylvania, USA
Key Words. Transcription factors • Spleen focus-forming virus • Hematopoietic stem cell • Multipotential progenitor cells • Gene targeting
Dr. Edward W. Scott, Institute for Human Gene Therapy, Stellar-Chance Laboratories, Room 401a, 422 Curie Boulevard, University of Pennsylvania, Philadelphia, PA 19104-6100, USA.
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Abstract |
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 Top Abstract IntroductionProbing PU.1 Function In...Functional Versatility of PU.1...Exploring PU.1 Function in...ConclusionReferences |
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Introduction |
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TopAbstractIntroductionProbing PU.1 Function In...Functional Versatility of PU.1...Exploring PU.1 Function in...ConclusionReferences |
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Numerous expression studies using both human and murine tumor-derived cell lines and tissue samples have shown expression of PU.1 to be restricted to hematopoietic cell lineages [3, 5-7]. PU.1 is expressed in the spleen, thymus, and bone marrow of mice, and both human and murine macrophage, mast cell, B lymphoid, proerythroblastic, and granulocytic cell lines. Promoter and enhancer dissection studies have localized a large number of potential PU.1-dependent regulatory elements to genes encoding components of the B cell receptor expressed by B lymphoid cells and an array of adhesion molecules, growth factor receptors, and lysozymal enzymes associated with myeloid cells. Biochemical and transactivation analyses have delineated discrete functional domains within the PU.1 protein. The domains include an N-terminal transactivation region, a downstream PEST domain, and a carboxy-terminal DNA-binding domain [3, 8-14].
To investigate the function of PU.1 in vivo, the PU.1 gene was mutated by gene targeting [15, 16]. The PU.1 mutation severely impaired the development of both the lymphoid and myeloid lineages. The effect on myeloid development was also substantiated by the inability of PU.1 mutant embryonic stem (ES) cells to differentiate into macrophages in vitro [17, 18]. These data not only confirmed a critical function for PU.1 during B lymphoid and myeloid development but also for the first time indicated that PU.1 is required for T cell development.
To initiate this review, an overview of the role of PU.1 in SFFV-induced erythroleukemias will be presented. This important biological system led to the original identification of PU.1 and indicated the importance of PU.1 regulation during erythropoiesis. In vivo studies using gene targeting will be extensively discussed to highlight PU.1-dependent events that occur during development of the lymphoid and myeloid lineages. In addition, data and working models will be presented to explain how the functional versatility of PU.1 may be achieved. The role of PU.1 in the regulation of genes required for proper development and functional integrity of both B lymphoid and myeloid cells will be discussed. The experimental inquiries summarized in this review should further develop our appreciation of the functional importance and uniqueness of PU.1 for the immune system.
Lessons Learned from Studying SFFV-Induced Erythroleumkias
Susceptible strains of mice can be infected with either the polycythemia (FLV-P)- or anemia (FLV-A)-inducing strains of the Friend virus, resulting in the development of fatal acute erythroleukemias within 8 to 14 weeks [19]. The FLV-P and FLV-A strains of Friend virus are a complex consisting of a replication-competent helper murine leukemia virus (MuLV) plus the spleen focus-forming virus (SFFV-P or SSFF-A, respectively). During the initial phase of the disease, a membrane glycoprotein, gp55, encoded by SSFV complexes with erythropoietin receptors (EpoR) to stimulate the proliferation of infected erythroblasts [20, 21]. During this polyclonal phase of the disease, the infected erythroblasts have a limited proliferative capacity and will undergo terminal differentiation into hemoglobinized cells [22]. The uncontrolled proliferative state observed during the initial phase of the disease has been attributed to the activation of both the Jak-Stat and Raf-1/MAP kinase signal transduction pathways [23].
After several weeks of infection, malignant proerythroblastic cells that are clonal or oligoclonal in nature begin to appear. These proerythroblastic leukemic cells have an unlimited proliferative capacity and do not differentiate further in vivo. Almost all Friend virus-induced proerythroleukemias studied thus far contain an SFFV provirus integrated 10 to 13 kb upstream of exon 1 of the PU.1 gene [1, 2, 24]. Integration of the SFFV into the PU.1 locus is thought to upregulate the expression of PU.1 due to the presence of the SSFV long terminal repeat functioning as a heterologous enhancer. The linkage between PU.1 and genesis of SFFV-induced erythroleukemias has been supported by work demonstrating that a PU.1-encoding retrovirus growth immortalizes murine bone marrow cells leading to the generation of erythroblastic cell lines [25]. More recently, an SFFV-LTR-driven PU.1 transgene has been reported to mimic several aspects of the Friend virus-induced erythroleukemias in transgenic mice [26]. A portion of the mice contain an Epo-dependent expansion of nonmalignant proerythroblastic cells that are partially blocked in differentiation and have upregulated PU.1. Epo-independent malignant erythroblastic cells arose later in these mice suggesting that additional genetic alterations are necessary for completion of the leukemogenic process. One potential secondary genetic change may involve the p53 gene, which is often mutated or becomes a site for proviral integration in SFFV-associated erythroleukemias [27, 28].
Murine erythroleukemia-derived cell lines (MEL) have been used as in vitro models to investigate the molecular events that occur during tumor progression. MEL cell lines are known to synthesize large amounts of PU.1 mRNA and protein [5, 24, 25, 29]. Treatment with dimethylsulfoxide (DMSO) or hemamethylene bisactamide (HMBA) causes the erythroleukemic cells to reenter the differentiation program, resulting in the rapid degradation of PU.1 mRNA and protein, and the upregulation of ß-globin gene expression [30-32]. The DMSO-mediated downregulation of PU.1 mRNA levels is due to a post-transcriptional event that is selective to PU.1 since other transcription factors, including GATA-1 and NF-E2, are unaffected [33].
The involvement of PU.1 in erythroid leukemogenesis is not a unique property of PU.1 since other members of the ETS family, namely v-ets and fli-1, have been implicated in erythroleukemic transformation [34-36]. Outside the hematopoietic system, PU.1 expression can initiate apoptotic cell death in fibroblasts [25]. Furthermore, apoptotic cell death can also be induced in differentiating erythroleukemic cells that overexpress PU.1 [37].
At present, the role of PU.1 during erythroid leukemogenesis is still not clear. It is believed that expression of PU.1 at nonphysiological levels adversely affects the function of regulatory proteins normally involved in erythroid differentiation. In this regard, a transcription factor regulatory hierarchy has been proposed from a recent study using MEL cells [32]. In their model, PU.1 is able to block differentiation of proerythroblasts in part by inducing the c-myb gene, which in turn is able to deregulate the expression of c-myc, and perhaps other genes. The crucial function of c-myb and c-myc in this leukemogenic pathway is evidenced by the ability of ecotopically expressed c-myc or c-myb to block HMBA-induced differentiation of MEL cells, even though PU.1 is downregulated. The requirement for downregulating c-myb during erythroid differentiation has been noted by several groups [38-40].
The large body of evidence presented thus far suggests the following scenario for the generation of SFFV-induced erythroleukemias. The combined action of Epo-driven expansion of erythroblasts and the PU.1-induced block in erythroblast differentiation, allows the accumulation of a large pool of proliferating immature erythrocytes which normally does not exist in uninfected animals. These abnormal conditions increase the frequency of an aberrant genetic event occurring, such as the inactivation of p53, resulting in the generation and outgrowth of one or more malignant erythroblastic clones.
The developmental stage at which SFFV-induced erythroleukemias arrest is strikingly similar to the stage at which GATA-1–/– proerythroblasts arrest [41, 42]. GATA-1–/– proerythroblasts express normal levels of EpoR and have upregulated several transcription factors, including GATA-2 and c-myb. These results further stress that the erythroblastic stage of erythropoiesis is a key regulatory checkpoint. For completion of the erythroid differentiation program, two regulatory events must be successfully completed. PU.1 expression must be downregulated, and GATA-1 expression must be maintained. A potential GATA-1 binding site has been identified in the proximal region of the PU.1 promoter region, which suggests that GATA-1 may be involved in the downregulation of PU.1 expression during terminal differentiation of erythrocytes [43]. However, in vitro transactivation studies have yet to demonstrate that GATA-1 is a negative regulator of the PU.1 promoter.
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Probing PU.1 Function In Vivo |
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TopAbstractIntroductionProbing PU.1 Function In...Functional Versatility of PU.1...Exploring PU.1 Function in...ConclusionReferences |
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Transcriptional Regulators of Fetal Hematopoiesis
Hematopoiesis is a dynamic developmental process that ensures a sufficient supply of terminally differentiated blood cells for the survival of the animal. The mature blood cells include erythrocytes, megakaryocytes, monocytes and lymphocytes, all originating from a population of self-renewing pluripotent stem cells (HSCs). Downstream of the HSC lies a series of multipotential progenitor populations with restricted differentiative potential, that eventually become committed to a defined blood cell lineage. The site of hematopoiesis changes during development [44]. During embryogenesis, primitive hematopoiesis is initiated in the yolk sac between days 7 and 8 with the production of nucleated erythrocytes followed by primitive macrophages, megakaryocytes, and multipotential progenitors. By day 9, the hematopoietic system shifts to the fetal liver where definitive hematopoiesis begins. This is followed by a final shift to the bone marrow, which is the primary site of hematopoiesis in the adult.
The generation and subsequent characterization of transcription-factor-deficient "knockout" animals has revealed that a number of transcription factors are required by the developing hematopoietic system. Those transcription factors that are absolutely required during yolk sac and fetal liver hematopoiesis are displayed in the form of a transcription factor hierarchy in Figure 1. Transcription factors rbt2/LMO2 and SCL/tal-1 are thought to directly impinge on the HSC since embryos deficient in either rbt2 or SCL have no detectable yolk sac or fetal liver hematopoiesis [45-47]. It is not known if either mutation affects hematopoietic commitment from the putative hemangioblast, or the differentiating potential of the HSC itself. The hemangioblast is the proposed common precursor for the hematopoietic and endothelial cell lineages.
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2 (CBF2) have a similar phenotype, but live a day or two longer than GATA-2–/– embryos. Both AML and GATA-2 mutants die of a severe anemia and contain little or no erythoid or myeloid progenitors [49, 50]. However, for both mutations, morphologically normal nucleated primitive erythrocytes were found in the yolk sac. In the case of the AML1 mutation, fetal livers from day 11.5 mutant embryos could be examined, and no definitive hematopoietic progenitors were detected [49]. Thus, both GATA-2 and AML1 appear to be required for expansion of primitive multipotent progenitors in the yolk sac. Targeting of the core-binding factor ß (CBFß) gene, which encodes the heterodimeric binding partner of AML1/CBF2, results in a similar detrimental effect on primitive hematopoiesis but a less severe effect on definitive hematopoiesis [51, 52]. In CBFß–/– fetal livers there are several hundred-fold fewer erythroid and myeloid progenitors. The behavior of the CBFß mutation implies a role in the expansion of both primitive and definitive multipotential progenitors. The c-myb mutation had no effect on the number of megakaryocytes, but exhibited a profound decrease of erythroid and myeloid progenitors [53]. In all these mutations, lymphoid progenitors were not examined, but are presumably missing.
There is an additional group of transcription factor mutations whose effect is restricted to a single lineage. This group includes GATA-1, EKLF, and C/EBP. Both GATA-1 and EKLF affect definitive erythropoiesis. GATA-1 mutant erythroid cells are blocked at the erythroblastic stage of differentiation, while EKLF affects ß-globin expression and therefore affects terminal differentiation [54, 55]. The C/EBP mutation resulted in a complete block in granulocyte terminal differentiation [42, 54-57].
PU.1 Function Elucidated from PU.1 Knockout Animals
To definitively understand the function of PU.1 during hematopoiesis, gene targeting of the PU.1 loci in ES cells was undertaken [15]. The ETS DNA-binding domain of PU.1, spanning amino acids 167 to 255, is composed of three -helices and a four-stranded anti-parallel ß sheet [58, 59] (Fig. 2). PU.1 binds DNA via a "winged" helix-turn-helix (loop-helix-loop) structure. Recognition of the PU box in the major groove is made via the 3 recognition helix while the two loop-like structures on either side contact the phosphate backbone in the minor groove. The first loop consists of ß-strands 3 and 4 (the wing), while the second is formed by the turn between helixes 2 and 3. The targeting strategy taken by Scott et al. is presented in the upper portion of Figure 2C. Amino acids 200 to 272, containing the entire loop-helix-loop recognition structure of PU.1 were deleted and replaced with a neomycin drug-selectable cassette. Deletion of the coding sequence for the loop-helix-loop structure ensured that no functional or partially functional PU.1 protein could be produced from the targeted allele.
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The primary focus of the initial analysis was to examine the developmental status of lymphoid and myeloid populations in the fetal liver and thymus. Histological staining for lysozyme and myeloperoxidase, coupled with flow cytometric staining (FACS) for the myeloid-specific surface markers CD11b/CD18 and Gr-1, indicated that myelopoiesis was blocked at a very early stage in PU.1–/– fetal livers. FACS analysis for the B lymphoid marker B220 combined with reverse transcriptase-PCR (RT-PCR) to detect Ig gene arrangement events yielded no detectable B lymphoid progenitors. Likewise, T cell progenitor populations expressing T lymphoid markers Thy1, CD2, CD4, and CD8 were absent in mutant fetal thymi.
Subsequent in vitro colony-forming unit (CFU) assays on day 16 fetal liver cells confirmed the complete lack of myeloid progenitors (CFU-G, CFU-M, CFU-GM, CFU-GEMM), but normal numbers of erythroid (CFU-E) and megakaryocyte (CFU-EMeg, CFU-Meg) progenitors in PU.1–/– embryos [15, 63]. Similar results were obtained for day 8.5 and 10.5 yolk sacs, which represent earlier sites of hematopoiesis [17]. This indicates that a similar block in myeloid differentiation occurs during yolk sac hematopoiesis. There was no discernible effect on the development of other major organs or tissues, which is consistent with the known expression pattern of PU.1. These initial results led to the conclusion that PU.1 may be required for lymphoid and myeloid commitment during fetal hematopoiesis. Furthermore, this body of evidence suggested the existence of a PU.1-dependent multipotential progenitor that gives rise to both the lymphoid and myeloid lineages. Progenitors with both myeloid and lymphoid differentiating potential in the fetal liver and the yolk sac have previously been described through the use of in vitro clonogenic assays [64, 65]. At present it is still not possible to rule out a separate requirement for PU.1 in committed progenitors from either the B lymphoid or myeloid lineages.
Several aspects of the PU.1–/– animals were unexpected. The very early nature of the defect would not have been predicted based on the known expression pattern of PU.1. Given that PU.1 is only expressed at high levels in the myeloid and B lineages, we would have expected a discernible block in development after commitment to these lineages. The absence of any detectable T cell progenitors was difficult to predict due to the controversial nature of PU.1 expression in the T cell lineage. PU.1 RNA expression has been detected in the adult thymus [3]. This expression may be due to an early T cell progenitor or a non-T cell component such as dendritic cells or macrophages [66]. PU.1 expression has been reported in one pro-T cell line, but expression in normal T cells is still unclear [66].
Two different experimental approaches were employed to further investigate the nature of the PU.1 mutation. The first approach involved the generation of ES-cell-derived chimeric animals, while the second employed the adoptive transfer of PU.1–/– fetal liver cells into lethally irradiated adult recipients [63]. Both strategies are designed to test the cell-autonomous nature of the PU.1 mutation by examining the functional behavior of PU.1–/– hematopoietic cells in a wild-type microenvironment.
Wild-type and PU.1–/– ES cells were used to generate chimeric animals with 50%-70% ES cell contribution based on coat color. There was no difference in contribution to non-hematopoietic tissues between PU.1+/+ and PU.1–/– ES cells. However, PU.1–/– ES cells could not contribute to any hematopoietic lineages in adult chimeras. The lack of erythroid contribution in the adult chimeras was puzzling because normal numbers of erythroid progenitors can be found in PU.1–/– fetal livers. As expected, a significant contribution of PU.1–/– ES cells to erythropoiesis was detected in day 16 fetal chimeras.
A similar phenomenon was observed when PU.1–/– fetal liver cells were adoptively transferred to lethally irradiated adult hosts. PU.1–/– fetal liver cells were unable to provide long-term radioprotection to the recipient animals, with death ensuing two to three weeks after irradiation. The PU.1–/– fetal liver cells did reconstitute the erythroid lineage for up to three weeks, but not the lymphoid or myeloid lineages. This is consistent with a fetal liver progenitor population being able to initiate erythropoiesis but unable to thrive in the bone marrow microenvironment. These data collectively indicate the cell-intrinsic nature of the PU.1 mutation which cannot be corrected by extracellular factors provided by a wild-type microenvironment.
The lack of contribution to erythropoiesis in adult chimeras and the failure of PU.1–/– fetal liver cells to either reconstitute or radioprotect adult animals suggest that there may be differing requirements for PU.1 in fetal versus adult hematopoiesis. Subsequent competitive reconstitution assays have determined that PU.1–/– fetal liver cells are able to contribute to erythropoiesis for only one month (J. Lovelock and E. Scott, unpublished results). This observation would suggest that PU.1–/– fetal HSCs are able to engraft in the bone marrrow but are at a competitive disadvantage compared to PU.1+/+ adult HSCs.
The inability of PU.1–/– fetal liver cells to contribute to either the myeloid or lymphoid compartments suggests a PU.1-dependent multipotent progenitor population that gives rise to both lineages in the fetal liver. This conclusion is supported by the observation that there is a four- to fivefold reduction in the AA4.1+/Lin– population found in PU.1–/– fetal livers. This fetal liver population contains a mixture of HSCs and multipotent progenitor cells, including a multipotential progenitor that can give rise to both B lymphoid and myeloid cells in vitro [67-69].
A second targeted disruption of the PU.1 locus has recently been reported [16]. This targeting approach inserted a neomycin resistance cassette at amino acid 232 located in the middle of the 3 recognition helix (lower portion of Fig. 2C). In contrast to the initial strategy's process, no PU.1 coding sequence was deleted. In addition, a different version of the neomycin resistance gene was employed with direction of transcription in the opposite direction. For the sake of clarity, the second targeted allele of PU.1 will be designated PU.1–/–(2) [16]. Likewise, references to the original PU.1 targeting will have the designation PU.1–/–(1) [15]. In contrast to the embryonic lethal phenotype of PU.1–/–(1), the second targeting strategy resulted in PU.1–/–(2) pups being born with the expected Mendelian frequency but succumbing to septicemia within 48 h. There was little or no effect on either the erythroid or megakaryocyte lineages, as expected. Analysis of the myeloid and lymphoid compartments in PU.1–/–(2) neonates yielded significant differences with PU.1–/–(1). PU.1–/–(2) neonates lacked Gr-1+ and F4/80+ myeloid cells in the bone marrow and liver which indicated the absence of mature granulocytes and macrophages. However, there was an abnormally large population of Mac-1+ cells, which may represent an accumulation of immature myeloid cells. PU.1–/–(2) neonates contain an aberrant population of B220+ cells in the bone marrow and spleen. The B220+ cells were BP-1+, HSA+, and CD43–. This phenotype does not correspond with any known B cell lineage, suggesting that B lymphopoiesis was severely disrupted by the PU.1–/–(2) mutation. Neonates also lacked CD4+/CD8+ thymocytes in the thymus.
Survival of the PU.1–/–(2) pups could be extended up to two weeks with antibiotic therapy. These older animals exhibited a delayed onset of T cell development, but near-normal levels of CD4+, CD8+, and CD4+/CD8+ thymocytes were eventually detected in the thymus. The aberrant B220+ cells in these mice failed to complete a normal B cell, which would suggest a very early block in B cell differentiation, perhaps at the pre-pro-B cell stage [70]. Normal myeloid differentiation is also affected in PU.1–/–(2) mice. There is the rapid appearance of an abnormally large population of Mac-1+ cells and the delayed accumulation of a Gr-1+ population. The presence of these immature myeloid progenitors, along with a very small number of mature granulocytes (Mac-1+/Gr-1+ or CAE+) and macrophages (F4/80+) in the spleen and bone marrow, is consistent with a severe impairment of myeloid development. Further examination of the bone marrow of these PU.1–/–(2) animals also demonstrated that they suffer from osteopetrosis due to a lack of osteoclast formation [71]. This finding was not unexpected since osteoclasts are derived from myeloid progenitors.
Based on the data describing the hematopoietic defect in the PU.1–/–(2) animals, one can conclude that PU.1 is necessary at an early stage in T cell development, perhaps at the point that multipotential progenitors become committed to the T cell lineage. The latter stages in T cell differentiation do not appear to be severely affected by PU.1–/–(2) mutation. Similarly, PU.1 is required early during B differentiation. The absence of PU.1 results in a total block during the pre-pro stage of B cell development. There is a differential effect of the PU.1–/–(2) mutation on myeloid development as evidenced by the differing rate of accumulation of immature monocytic and granulocytic progenitors. Terminal differentiation of both granulocytes and macrophages was not totally blocked, but was severely impaired. There appeared to be no significant effect on commitment to either the B lymphoid or myeloid lineage.
McKercher et al. have proposed several explanations for the observed phenotypic differences between the two PU.1 mutations, including: differences in ES cells used, targeting scheme, and the neomycin transcriptional cassette. The authors concluded that the most likely explanation was our use of the strong phosphoglycerate kinase (PGK) promoter regulating the neomycin resistance gene in the same orientation as the PU.1 gene [15] (Fig. 2C). It was postulated that the severity of the PU.1–/–(1) mutation was due to the effects of the PGK promoter on a downstream gene(s) involved in both lymphoid and myeloid differentiation. The ability of a PU.1 transgene to rescue myeloid differentiation of PU.1–/–(1)–ES cells would suggest that this explanation is highly unlikely [17]. This demonstrates that the observed phenotype for the PU.1–/–(1) mutation is the direct result of the lack of PU.1 expression [17]. A more likely explanation for the differences between the two PU.1 knockout phenotypes is that PU.1–/–(2) mice may express a truncated PU.1 protein that retains limited functional ability. This prediction is consistent with a less severe form of the PU.1–/–(1) phenotype seen in the PU.1–/–(2) mice. It has been demonstrated that the PU.1–/–(1) mutation results in no detectable levels of PU.1 protein in either the fetal liver or thymus [63]. RT-PCR analysis has also been used to determine that no truncated PU.1 transcripts are expressed in PU.1–/–(1) fetal livers. A comparable type of analysis of PU.1 protein and RNA expression has not been reported for PU.1–/–(2) animals.
In summary, the studies completed thus far on the two different PU.1 mutations have allowed further speculation concerning the requirements for PU.1 during hematopoietic differentiation. As depicted in Figure 1, low levels of PU.1 would be required for commitment to the lymphoid and myeloid lineages. The lack of expression of PU.1 in most T cell lines would suggest that once lymphoid progenitors are committed to the T lineage, further differentiation does not require PU.1 [3, 6, 66]. This would enable a small population of cells committed to the T cell lineage to expand and differentiate into mature T cells in the absence of a fully functional PU.1 protein. Such a scenario may explain the observed delay in T cell development in PU.1–/–(2) neonates [14]. The detection of both immature B cell and myeloid progenitors is consistent with PU.1–/–(2) animals producing a truncated PU.1 protein that retains some functional ability. Sufficient levels of PU.1–/–(2) protein may be produced to overcome the block in lymphoid and myeloid commitment that is observed in PU.1–/–(1) embryos [15, 63]. The severe impairment of myeloid development and a complete block in B cell differentiation in PU.1–/–(2) neonates may be due to insufficient levels or function of the PU.1–/–(2) protein. This model is supported by the fact that both murine multipotential progenitors and ES cells upregulate PU.1 as the cells differentiate along the myeloid pathway [17, 72].
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Functional Versatility of PU.1 during Hematopoiesis |
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TopAbstractIntroductionProbing PU.1 Function In...Functional Versatility of PU.1...Exploring PU.1 Function in...ConclusionReferences |
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Promoters
In the myeloid lineage, a large collection of PU.1-dependent promoters have been identified that control an array of genes encoding predominantly growth factor receptors and adhesion molecules. The collection includes the high-affinity FcI/CD64 [73, 74] and low-affinity FcIIIA/CD16 receptors [75]; the receptors for G-CSF [76], GM-CSF [77], and the colony-stimulating factor (CSF/c-fms) [78]; proteinase-3 [79]; the adhesion molecules CD11b [80] and CD18 [81, 82]; the scavenger receptor A gene [83] and the tyrosine kinase c-fes/c-fps [84, 85]. For the B cell lineage, the promoter region of the J chain gene has been shown be PU.1-dependent [12].
The majority of these PU.1-dependent promoters in the myeloid lineage share several structural features, including a PU.1 binding site close to the site(s) of transcriptional initiation, no TATA box, and a cluster of binding sites for Sp1, members of the CCAAT/enhancer binding protein (C/EBP) or core binding protein (CBF) families [86]. A schematic layout of the transcription factors binding to the proximal promoter regions of M-CSFR, GM-CSFR, and CD11b is shown in Figure 3. In contrast to PU.1, the other transcription factors are expressed widely outside the hematopoietic system, thereby indicating that myeloid-specific expression may be principally due to PU.1 or the unique combination of adjoining transcription factors working in concert with PU.1. In this group of myeloid promoters, the binding of PU.1 close to the start(s) of transcription may facilitate the recruitment of the TFIID complex and promote the binding of the adjoining transcription factors. TFIID is a protein complex consisting of TATA-binding protein (TBP) and a set of TBP-associated factors (TAFs) [87]. Binding of TFIID to the promoter region is the first step of the assembly process that forms the transcription initiation complex. Activating transcription factors such as PU.1 usually make contact with TFIID via an interaction with a TAF. However, the N-terminal transactivating portion of PU.1 has been shown to bind TBP, suggesting a direct interaction between PU.1 and TBP [10]. Even though PU.1 may make the initial contact with TFIID, downstream transcriptional factors are likely to help stabilize the TFIID complex through TAF interactions. The PU.1-initiated cooperative stabilization of TFIID may be sufficient to evoke myeloid-specific expression for this group of genes.
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As shown in Figure 4 for the Ig kappa 3' enhancer region, binding of PU.1 to its site recruits the transcription factor Pip (PU.1 interacting partner), followed by the binding of c-fos and c-jun to form a cooperative higher-order enhancer complex that synergistically activates transcription in vitro [90] (Fig. 4). Similarly, binding of PU.1 and ETS-1 to adjoining sites in the heavy chain intron enhancer region results in a protein:protein interaction that promotes the assembly of a multiprotein complex with the downstream basic helix-loop-helix-leucine zipper family member, TFE3, for maximal in vitro enhancer activity [91-93]. A PU.1 mutant lacking the entire transactivation domain is able to synergistically activate either the Ig kappa 3' enhancer or the heavy chain intron enhancer [90, 91]. These experiments indicate that the interaction between PU.1 and the other transcription factors bound to either the Ig heavy or light chain enhancer region generates a regulatory complex that is not dependent on the transactivating domains of PU.1. For these enhancer regions, the crucial function of PU.1 is to serve as an architectural transcription factor. In this capacity, the binding of PU.1 induces bending of DNA that promotes the binding of other transcription factors to nearby regulatory elements, thus forming a higher-order protein-enhancer complex. The PU.1-induced bending of DNA and the subsequent protein:protein interaction between other components of the multiprotein-enhancer complexes repositions the transactivation region of one or more of the adjoining transcription factors to augment transcription.
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Exploring PU.1 Function in an ES cell In Vitro Differentiation System |
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TopAbstractIntroductionProbing PU.1 Function In...Functional Versatility of PU.1...Exploring PU.1 Function in...ConclusionReferences |
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PU.1–/– ES cells fail to differentiate into macrophages in vitro [17, 18]. The failure of PU.1–/– ES cells to differentiate into macrophages can be rescued by a PU.1 transgene under the control of its own promoter [17]. This result confirms the pivotal role that PU.1 serves in controlling macrophage differentiation. The ability to rescue macrophage differentiation by re-expressing PU.1 through an introduced transgene has allowed the function of previously described PU.1 domains to be reexamined. In this experimental system, a genetic approach was taken to test a panel of PU.1 mutants and determine their effect on the development of normal mature macrophages in vitro. The successful generation of mature macrophages was monitored by expression of the myeloid surface markers CD11b and F4/80.
Conventional biochemical and transactivation assays using both tumor-derived B cell and non-hematopoietic cell lines have identified a number of functionally important regions of the PU.1 protein. Multiple transactivation domains in the N-terminus of the protein have been identified (amino acids 7 to 100; [10, 12-14]). Three domains are rich in acidic amino acids (amino acids 7 to 74) while a third is glutamine-rich (amino acids 75-100). Of the four transactivation regions identified, the glutamine-rich domain appears to be the weakest [14]. One of the acidic activation domains contains two serine phosphorylation sites located at amino acids 41 and 45 that have been determined to be necessary for promoting M-CSF-dependent proliferation of bone marrow macrophages [100].
Downstream of the transactivation region is the PEST domain (amino acids 118 to 160), which is rich in proline, glutamic acid, serine, and threonine residues. The PEST domain is not required for transactivation as measured by transient transfection assays in non-hematopoietic cell lines [13, 14, 91]. However, a phosphorylation site at serine 148 of the PEST domain mediates the interaction between PU.1 and Pip in vitro [9, 11]. The interaction between the transcription factor Pip and PU.1 is required for optimal in vitro transactivation of 3' enhancer regulatory elements located in the kappa and lambda immunoglobulin light chain genes in non-hematopoietic cell lines [11, 101]. Reduced serum immunoglobulin levels and impaired humoral responses in Pip-deficient mice substantiate the crucial nature of the interaction between PU.1 and Pip for the B lymphoid lineage in vivo [102].
DNA-binding activity resides in the 85 amino acids long ETS domain located in the carboxy-terminus of PU.1 [3, 8]. PU.1 binds as a monomer to its DNA-binding site through a "loop-helix-loop" structure resulting in DNA bending [58]. Recently, a second transcription factor, NF-IL6ß (C/EBP
), has been found to interact directly with PU.1 and modulate the transcriptional activity of PU.1 in vitro [103]. The binding site for NF-IL6ß maps to the C-terminal 28 amino acids of PU.1.
Utilizing the ES cell in vitro differentiation system, multiple regions of the PU.1 protein were determined to be required for ES cells to differentiate into CD-11b+ and F4/80+ macrophages. In addition to the DNA-binding domain, a subregion of the PEST domain was determined to be required for the differentiation of CD-11b+ and F4/80+ macrophages from ES cells. This does not involve the previously identified serine 148 residue, which is important for controlling gene expression in the B lineage, implying that a novel protein:protein interaction is occurring through the PEST domain which is absolutely necessary for myelopoiesis. Neither of the acidic transactivation regions had a role in myeloid development. Instead, the glutamine-rich region, previously described as a weak transactivation region in vitro [14], was absolutely required for myeloid differentiation. Additional mutations affecting serine phosphorylation sites 41, 45, or 148 or deleting the C/EBP binding site had no effect on myelopoiesis (R.Fisher and E. Scott, unpublished results).
The results of this current study create a conundrum concerning the functional importance of acidic activation domains for PU.1 function. Numerous studies have demonstrated potent transactivation potential for the acidic activation domains of PU.1 using non-hematopoietic cells and a classical transactivation assay system [10, 13, 14]. There are several possible explanations that could rectify this discrepancy. The acid domain of PU.1 may play a specialized role in controlling PU.1-dependent enhancers. The lack of an acidic domain would have no effect on macrophage development because most PU.1-dependent myeloid genes are regulated through their promoters. In vitro studies of the Ig heavy chain intron enhancer and the Ig kappa and lambda light chain 3' enhancer presented earlier in this review do not support this possibility. In both studies, a PU.1 mutant lacking the acidic domains was able to promote enhancer function [90, 91]. The use of non-hematopoietic cells to measure PU.1 transactivation activity may be a more likely explanation for over-emphasis of the importance of the acidic domains. The acidic domains may function better to activate reporter constructs in an abnormal cellular milieu. Indeed, Shin and Koshland used a terminally differentiated B cell line to assess PU.1's transactivation potential and concluded that transactivation activity only resided in the glutamine-rich domain [12]. The functional importance of the glutamine-rich transactivation domain and the PEST domain for PU.1 function can best be verified by introducing the desired mutants into the germline of mice and testing their effect on lymphoid and myeloid cells in vivo.
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-deficient mice. Proc Natl Acad Sci USA 1997;94:569-574.
) activation of the human high-affinity Fcg receptor gene: a novel IFN regulatory mechanism. Mol Cell Biol 1993;13:2182-2192.
