Transcription factors in hematopoiesis

575 Transcription factors in hematopoiesis Isaac Engel* and Cornelis Murre† The advent of gene targeting in the mouse has led to rapid advances in the identification of factors controlling gene expression that are essential for normal hematopoietic development. Recent work has also uncovered roles for some of these factors in leukemogenesis and in the global regulation of chromatin structure. Addresses Department of Biology, University of California at San Diego, La Jolla, California 92093-0366, USA *e-mail: [email protected] † e-mail: [email protected] Current Opinion in Genetics & Development 1999, 9:575–579 0959-437X/99/$ — see front matter © 1999 Elsevier Science Ltd. All rights reserved. Abbreviations bHLH basic helix-loop-helix C/EBP CCAAT/enhancer binding protein DN double negative EBF early B-cell factor ES embryonic stem FTOC fetal thymic organ culture Hes1 Hairy/Enhancer of split 1 HSC hematopoietic stem cell IL interleukin NK natural killer T-ALL T-cell acute cell lymphoblastic leukemia TCR T-cell receptor Introduction The nineties have been a very good decade for research in hematopoietic development — particularly with respect to the identification of transcription factors that play critical roles in the development of the various lineages that descend from the hematopoietic stem cell (HSC). Clearly, the critical element that has accelerated progress in these areas has been the ability to manipulate the mouse genome via homologous recombination into embryonic stem (ES) cells. The success of this approach in identifying transcription factors with essential roles in hematopoiesis has expanded our knowledge to the extent that a comprehensive review is beyond the scope of this article. Instead, we discuss a few of the areas in which recent progress has been made in further defining the roles of several transcription factors in hematopoiesis. Studies in lineage commitment The ability of gene targeting to reveal requirements for a protein in lineage commitment is often limited by a lack of phenotypic markers for the earliest committed progenitor cells. One approach that has been used to address the role of specific transcription factors in lineage commitment is through the enforced expression of these factors in cells that have at least some capability to differentiate. One such system that has been described employs of primary transformed multipotent progenitors generated by transducing chicken blastoderm cells with the E26 leukemia virus [1]. Recently, the effects of transduction of an inducible form of the Ets family transcription factor PU.1 into these cultures was examined [2••]. Whereas long-term expression of PU.1 resulted in myeloid commitment, short-term induction of PU.1 activity led to the formation of immature eosinophils. These data were consistent with gene-targeting experiments showing PU.1 to be required for myeloid development and also suggested the existence of a bilineage intermediate capable of progressing down either myeloid or eosinophil developmental pathways [3–5]. In parallel studies involving the transduction of α and β isoforms of CCAAT/enhancer binding protein (C/EBP), it was demonstrated that these transcription factors could induce the commitment and maturation of eosinophils from multi-potential progenitors, although the β isoform could also induce myeloid differentiation [6••]. Commitment to T- and NK-cell lineages has been examined by retroviral transduction into various purified human multi-potential populations followed by fetal thymic organ culture (FTOC). It was demonstrated that forced expression of Id3 — a dominant negative inhibitor of bHLH transcription factors such as those encoded by E2A and HEB — in multipotential progenitors blocks T-cell development and results in the increased production of NK cells [7]. Subsequently, analogous experiments were reported showing that Id3 also inhibits committed T-cell precursors from developing into TCRαβ T-cells, but does not affect TCRγδ T-cell development [8•]. These data are essentially consistent with the phenotypes observed in mice deficient for E2A and HEB, and suggest that bHLH activity may determine thymocyte developmental fates at multiple stages of differentiation. It should be noted, however, that E2A-deficient mice have been reported to have reduced numbers of adult, though not fetal, γδ T-cells [9•]. An additional study employing transduced human thymocytes in FTOC demonstrated that blocking of the IL-7 receptor/STAT 5 signaling pathway in pre-T cells also inhibited differentiation [10••]. Another enforced expression approach has been used to distinguish between the roles of E2A and early B-cell factor (EBF) in the differentiation of B-cells. Targeted mutations of the genes encoding these transcription factors have demonstrated that they are both required for progression past a very early stage in B-cell development [11–13] but the reported phenotypes of the knockout mice for these genes failed to indicate clearly whether or not one of these factors acts upstream of the other. This issue was addressed by examining the effects of enforced expression of E12 — one of the two major products of the E2A gene — and EBF on the phenotype of a 70Z/3 macrophage 576 Differentiation and gene regulation line. It was found that E12 acted largely to convert this line to a pre-B phenotype, as determined by the expression of a number of markers, including EBF [14••]. Enforced EBF expression, however, activated only a subset of the pre-B specific genes induced by E12, and did not result in the induction of E2A. These data suggest that E2A functions upstream of EBF in B-cell development. Additional transcription factors controlling Tand NK-cell development Embryonic lethality is a frequent consequence of the inactivation of transcription factors that are important in hematopoiesis, and often precludes a direct examination of their effect on relatively late developmental stages. Such studies, however, can often be performed by generating chimeric mice whereby ES cells with lethal mutations are implanted into wild-type or Rag1- or 2-deficient blastocysts. The contribution of the ES cells to various lineages can be determined by using allotypic differences to distinguish mutant from wild-type-derived tissue. Rag-deficient blastocysts are frequently used in these studies to block antigen receptor rearrangement, and hence lymphocyte development, in the host tissue. Two reports employing such an approach have recently been published that revealed roles for the bHLH gene Hes1 (Hairy/Enhancer of split 1) and the c-myb proto-oncogene in T-lymphocyte development. Hes1 is a target of the Notch pathway that can function as a transcriptional repressor, and is known to be required for proper nervous system development [15–17]. Hes1-deficient thymocytes were unable to progress through the CD4/CD8 double negative (DN) stage of development, during which TCR rearrangements are initiated [18••]. Another recent report demonstrated that c-myb, in addition to being essential for fetal erythropoiesis, was also needed for T-cell development [19,20••]. c-Myb-deficient thymocytes were arrested at a very early developmental stage, prior to the expression of either the CD44 or CD25 markers that are frequently used to characterize DN thymocytes. Furthermore, it was determined that c-myb –/– ES cells were also unable to contribute to either the B-cell or macrophage compartments. Two new mutations affecting NK-cell development have also been reported recently. Ets-1-deficient mice were found to have greatly reduced numbers of NK-cells and no detectable NK response either in vitro or in vivo [21••]. Mice lacking the bHLH-inhibitor protein Id2 were also found to have reductions in NK-cell number and in vitro activity, which is consistent with the findings described above that bHLH activity controls the T/NK lineage decision [7,22•]. SCL and LMO proteins in normal and aberrant development The SCL/Tal1 gene (SCL) encodes a type II bHLH protein that can bind E box DNA sequences by forming heterodimers with type I bHLH proteins encoded by the E2A, HEB or E2-2 genes [23,24]. Targeted mutations in SCL revealed that this gene is essential for yolk-sac erythropoiesis, and SCL null ES cells fail to contribute to any hematopoietic lineages in chimeric mice [25]. Furthermore, studies with erythroleukemia lines support a role for SCL in erythroid development [26]. Recent data suggest that SCL is required for vascular differentiation as well. One such line of evidence comes from an attempt to correct the SCL null phenotype with a transgenic construct consisting of an SCL cDNA under control of a promoter that allows for expression in HSCs. Although the transgene was found to rescue yolk-sac hematopoiesis, the resulting embryos were not viable due to defects in blood vessel formation [27••]. Thus SCL has dual roles in hematopoiesis and angiogenesis. In addition, studies in zebrafish have provided evidence for a role for SCL in the development of a presumptive hemangioblast. One study demonstrated that overexpression of SCL in zebrafish embryos resulted in overproduction of both hematopoietic and endothelial precursors, whereas another group found that SCL acts downstream of the zebrafish gene cloche to specify both hematopoietic and vascular differentiation [28••,29••]. LMO2/RBTN-2 (LMO2), a member of the LIM-only domain family, has been shown to interact with SCL, and LMO2–/– embryos display hematopoietic defects similar to that of embryos with null mutations in SCL [30,31•]. Recently, the complexes containing SCL and LMO2 have been characterized in greater detail. SCL and LMO2 have been shown in erythroid cells to associate with E2A, GATA-1 and the LIM-binding protein Ldb1/NL1 in a complex that binds a DNA sequence consisting of E box and GATA sites separated by ~9 bp [32]. Recently, similar binding sites were immunoprecipitated from fragmented murine erythroleukemia chromatin using anti-SCL antibodies [33•]. One such site was found to be located within a gene whose expression correlated with that of SCL and GATA-1 in both transformed cell lines and an in vitro model system of erythroid differentiation. There have been a number of studies exploring the possibility of a relationship between the roles of SCL and LMO proteins in normal hematopoiesis and in T-cell acute lymphoblastic leukemia (T-ALL). A large fraction of T-ALL isolates are characterized by aberrantly high expression of SCL and either LMO1 or 2 [34,35]. SCL and LMO proteins have also been shown to collaborate to induce thymic lymphomas in mouse transgenic models [36]. A bindingsite selection protocol was used to demonstrate that SCL and LMO2 present in extracts from LMO2 transgenic Tlymphomas could interact to form DNA-binding complexes [37•]. Like the erythroid-specific complex described above, the complex found in T-lymphomas contained the E2A-encoded E47 protein along with Ldb1/NL1. It differed from the previously described complex, however, in that it did not include a GATA family member and assembled on a DNA binding site that consisted of two E boxes. Interestingly, this site was similar to one in the promoter of the c-kit gene, which was found by Transcription factors in hematopoiesis Engel and Murre Krosl et al. [38••] to be positively regulated by SCL in two hematopoietic cell lines. On the other hand, a group that identified retinaldehyde dehydrogenase 2 (RALDH2) as a target of SCL and LMO in human T-ALLs characterized a promoter in the RALDH2 gene that contained a GATA-3 binding site critical for SCL and LMO-dependent expression in T-ALLs [39•]. Although an E box was found adjacent to this GATA site, SCL binding to DNA was found to be dispensable for promoter activity. Taken together, it appears that there is considerable heterogeneity in the DNA-binding complexes that are formed by SCL and LMO proteins. Nevertheless, the reports of SCL/LMO complexes in T-lymphomas strengthen the contention that these proteins collaborate to induce oncogenic transformation, presumably by the inappropriate activation of critical target genes. There is an alternative way at looking at the contribution of SCL and LMO proteins to T-ALL, however. SCL has long been known to inhibit E2A activity, as SCL/E2A heterodimers have been shown to have greatly reduced transcriptional activity relative to that of E2A homodimers [40,41]. Mice with targeted mutations in the E2A locus are also highly susceptible to T lymphoma [42,43]. Furthermore, both E2A-deficient and SCL/LMO doubletransgenic thymocytes exhibit partial blocks during the DN stage of development prior to the onset of lymphoma, although the defects in these two genetic backgrounds differ in several respects [36]. Recently it has been shown that E2A activity can act to kill either the Jurkat T-ALL line — which expresses high levels of SCL and LMO1 — or murine T-lymphoma lines derived from E2A-deficient mice [44•,45•]. In addition, E2A activity was also found to inhibit cell-cycle progression in Jurkat cells [44•]. These data strongly imply that E2A acts a tumor suppressor in thymocytes, and further suggest a relationship between lymphomas induced by either ectopic expression of SCL and LMO or a deficiency in E2A activity. However, the question as to whether SCL and LMO proteins act to induce lymphoma through an inhibition of E2A activity has yet to be answered definitively. The Ikaros family in chromatin modulation Ikaros was originally identified as a gene encoding multiple forms of a zinc finger domain protein with potential for both transcriptional activation and repression [46]. Mice deficient in Ikaros activity exhibited a number of severe defects in the development of lymphoid lineage cells [46]. Another member of the Ikaros family, Aiolos, was recently shown to be required for regulating B-cell activation [47••]. It now appears, however, that the Ikaros family of factors may not function as ‘conventional’ transcription factors — that is, by activating or repressing the expression of a select group of target genes. This was first implied by the finding that Ikaros complexes were found to be associated with centromeric heterochromatin [48]. Recently it was demonstrated that Ikaros formed toroidal structures that colocalized with DNA replication centers in activated 577 T-cells, and that this property may be important for proper chromosome propogation in these cells as well as for setting general thresholds of activation [49••]. Another report showed that Ikaros and Aiolos were associated with large nuclear complexes that had histone deacetylase and chromatin remodeling activity [50•]. It will be interesting to see if analogous functions will eventually be ascribed to other hematopoietic transcription factors. Conclusion and future directions The quantum leap in the power of the mouse as a genetic tool through the advent of gene-targeting technology has allowed the identification of a number of transcription factors with critical roles in hematopoietic development. Certainly other similar factors remain to be discovered. In this regard, the development of the zebrafish as a research tool will faciliate the discovery of more hematopoietic regulators through forward genetic approaches. It could be argued, however, that at this point the largest gaps in our understanding of hematopoiesis lie in our knowledge of the critical genes that are regulated by these essential transcription factors. It is likely that the differential screening of arrayed cDNA libraries will be used increasingly to find these target genes. We also anticipate an increased use of retroviral transduction into primary cells as a technique to either ectopically express or repress particular transcription factors, both for the generation of sources of RNA for differential cloning and for lineage commitment studies. Two other areas of future research deserve mention. One concerns the effect that hematopoietic microenvironments have on gene expression. The importance of this question was demonstrated recently by the finding that globin expression patterns in fetal and adult erythocytes could be reprogrammed by transfer into the reciprocal microenvironment [51••]. Finally, the regulation of chromatin structure during hematopoiesis is just beginning to be explored. Although questions relating to chromatin modulation have not typically been the main focus of those interested in hematopoietic development, they are likely to become increasingly important. This is suggested by the recent data concerning Ikaros, as well as by recent reports of chromatin modulation upon the activation and differentiation of mature T-cells [52••,53•]. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1. 2. •• Graf T, Mcnagny K, Brady G, Frampton J: Chicken erythroid cells transformed by the gag-myb-ets-encoding E26 leukemia virus are multipotent. Cell 1992, 70:201-213. Nerlov C, Graf T: PU.1 induces myeloid lineage commitment in multipotent hematopoietic progenitors. Genes Dev 1998, 12:2403-2412. Nerlov and Graf have transduced multi-potential chicken cells with inducible forms of PU.1 to confirm a role for this protein in myeloid commitment. The authors also demonstrate that the transactivation domain of PU.1 is necessary for its function in biasing differentiation, and that transient expression of PU.1 can lead to an immature eosinophil-like phenotype. 578 Differentiation and gene regulation 3. Scott EW, Simon MC, Anastasi J, Singh H: Requirement of the transcription factor PU.1 in the development of multiple hematopoietic lineages. Science 1994, 265:1573-1577. 4. Scott EW, Fisher RC, Olson MC, Kehrli EW, Simon MC, Singh H: PU.1 functions in a cell-autonomous manner to control the differentiation of multipotential lymphoid-myeloid progenitors. Immunity 1997, 6:437-447. 5. McKercher S, Torbett B, Anderson K, Henkel G, Vestal D, Garibault H, Klemsz M, Feeney A, Wu G, Paige C, Maki R: Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J 1996, 15:5647-5658. 6. •• Nerlov C, McNagny KM, Doderlein G, Kowenz-Leutz E, Graf T: Distinct C/EBP functions are required for eosinophil lineage commitment and maturation. Genes Dev 1998, 12:2413-2423. Nerlov et al. studied the effect of C/EBP α and β isoforms on the differentiation of chicken multi-potential hematopoietic progenitors. They find that forced expression of both isoforms could induce eosinophil differentiation, and that C/EBPβ also induced myeloid differentiation. On the basis of the data presented in references [2••,6••], the authors propose a model in which PU.1 and C/EBP stimulate, and GATA-1 opposes, myeloid and eosinophil differentiation. 7. Heemskerk MHM, Blom B, Nolan G, Stegmann APA, Bakker AQ, Weijer K, Res PCM, Spits H: Inhibition of T cell and promotion of natural killer cell development by the dominant negative helix loop helix factor Id3. J Exp Med 1997, 186:1597-1602. 8. • Blom B, Heemskerk MHM, Verschuren MCM, van Dongen JJM, Stegmann APA, Bakker AQ, Couwenberg F, Res PCM, Spits H: Disruption of αβ but not γδ T cell development by overexpression of the helix-loop-helix protein Id3 in committed T cell progenitors. EMBO J 1999, 18:2793-2802. See annotation [9•]. 9. • Bain G, Romanow WJ, Albers K, Havran WL, Murre C: Positive and negative regulation of V(D)J recombination by the E2A proteins. J Exp Med 1999, 189:289-300. The Spits laboratory [7,8•] has developed a system whereby human progenitor cells at different stages of commitment to the T-cell lineage are transduced with recombinant retrovirus and then allowed to differentiate in FTOCs. They tested the effect of inhibition of bHLH transcription factors (by forced expression of Id3) on multi-potential progenitors isolated from human fetal liver and in committed ‘pre-T-cells’ obtained from human thymus samples [7,8•]. In the former case differentiation was skewed towards NK and away from T-fates, whereas in the latter case αβ T-cell development, but not γδ development was blocked. Thus T-cell development is sensitive to inhibitors of bHLH activity at multiple stages. Bain et al. characterized γδ development in E2A-deficient mice and found that the numbers of post-partum-type γδ T-cells were decreased, whereas fetal types were normal [9•]. 10. Pallard C, Stegmann APA, van Kleffens T, Smart F, Venkitaraman A, •• Spits H: Distinct roles of the phosphatidylinositol 3-kinase and STAT5 pathways in IL-7-mediated development of human thymocyte precursors. Immunity 1999, 10:525-535. Pallard et al. test the effect of inhibition of the IL7/STAT 5 signaling pathway, through forced expression of dominant negative forms of both STAT 5 and a chimeric IL-7 receptor on T-cell development, in their FTOC differentiation assay. Differentiation of αβ T-cells is blocked by the disruption of signaling through IL7/STAT 5. The authors also identify a role for signaling through phosphatidylinositol 3-kinase and protein kinase B in thymocyte proliferation and survival. 11. Bain G, Maandag E, Izon D, Amsen D, Kruisbeek A, Weintraub B, Krop I, Schlissel M, Feeney A, van Roon M et al.: E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements. Cell 1994, 79:885-892. 12. Zhuang Y, Soriano P, Weintraub H: The helix-loop-helix gene E2A is required for B cell formation. Cell 1994, 79:875-884. 13. Lin H, Grosschedl R: Failure of B-cell differentiation in mice lacking the transcription factor EBF. Nature 1995, 376:263-267. 14. Kee BL, Murre C: Induction of early B cell factor (EBF) and •• multiple B lineage genes by the basic helix-loop-helix transcription factor E12. J Exp Med 1998, 188:699-713. Kee and Murre provide evidence that E2A functions upstream of EBF on the basis of the ability of these factors to convert the 70Z/3 macrophage line to a pre-B-like phenotype. 15. Sasai Y, Kageyama R, Tagawa Y, Shigemoto R, Nakanishi S: Two mammalian helix-loop-helix factors structurally related to Drosophila hairy and Enhancer of split. Genes Dev 1992, 6:2620-2634. 16. Kim HK, Siu G: The notch pathway intermediate HES-1 silences CD4 gene expression. Mol Cell Biol 1998, 18:7166-7175. 17. Ishibashi M, Ang S-L, Shiota K, Nakanishi S, Kageyama R, Guillemot F: Targeted disruption of mammalian hairy and Enhancer of split homolog-1 (HES-1) leads to upregulation of neural helix-loophelix factors, premature neurogenesis, and severe neural tube defects. Genes Dev 1995, 9:3136-3148. 18. Tomita K, Hattori M, Nakamura E, Nakanishi S, Minato N, Kageyama R: •• The bHLH gene Hes1 is essential for expansion of early T cell precursors. Genes Dev 1999, 13:1203-1210. See annotation [20••]. 19. Mucenski ML, McLain K, Kier AB, Swerdlow SH, Schreiner CM, Miller TA, Pietryga DW, Scott JWJ, Potter SS: A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis. Cell 1991, 65:677-689. 20. Allen RD, Bender TP, Siu G: c-Myb is essential for early T cell •• development. Genes Dev 1999, 13:1073-1078. Roles for Hes-1 and c-Myb in T-cell differentiation were recently revealed through the analysis of chimeric mice [18••,20••]. Hes-1 was found to be required for the differentiation and expansion of DN thymocytes, whereas thymocytes deficient for c-Myb express markers arrested at the earliest defined stage of T-cell commitment. The c-Myb-null ES cells also failed to contribute to the B-cell or macrophage lineages. 21. Barton K, Muthusamy N, Fischer C, Ting C-N, Walunas TL, Lanier LL, •• Leiden JM: The Ets-1 transcription factor is required for the development of natural killer cells in mice. Immunity 1998, 9:555-563. See annotation [22•]. 22. Yokota Y, Mansouri A, Mori S, Sugawara S, Adachi S, Nishikawa S-I, • Gruss P: Development of peripheral lymphoid organs and natural killer cells depends on the helix-loop-helix inhibitor Id2. Nature 1999, 397:702-706. These papers [21••,22•] demonstrate the requirement for Ets-1 and Id2 in the development of NK cells. Mice deficient in Id2 were also found to lack lymph nodes and Peyer’s patches. 23. Voronova AF, Lee F: The E2A and tal-1 helix-loop-helix proteins associate in vivo and are modulated by Id proteins during interleukin-6 induced myeloid differentiation. Proc Natl Acad Sci USA 1994, 91:5952-5956. 24. Hsu H-L, Cheng JT, Chen Q, Baer R: Enhancer-binding activity of the tal-1 oncoprotein in association with the E47/E12 helix-loophelix proteins. Mol Cell Biol 1991, 11:3037-3042. 25. Orkin SH, Zon LI: Genetics of erythropoiesis: induced mutations in mice and zebrafish. Annu Rev Genet 1997, 31:33-60. 26. Aplan PD, Nakahara K, Orkin SH, Kirsch IR: The SCL gene product: a positive regulator of erythroid differentiation. EMBO J 1992, 11:4073-4081. 27. •• Visvader JE, Fujiwara Y, Orkin SH: Unsuspected role for the T-cell leukemia protein SCL/tal-1 in vascular development. Genes Dev 1998, 12:473-479. Visvader et al. find that although the early hematopoietic defect of SCL could be rescued by a transgene that placed a SCL cDNA under control of the GATA-1 promoter, vitelline vessel formation was defective and the embryos died by E9.5. 28. Gering M, Rodaway ARF, Gottgens B, Patient RK, Green AR: The •• SCL gene specifies haemangioblast development from early mesoderm. EMBO J 1998, 17:4029-4045. See annotation [29••]. 29. Liao EC, Paw BH, Oates AC, Pratt SJ, Postlethwait JH, Zon LI: •• SCL/Tal-1 transcription factor acts downstream of cloche to specify hematopoietic and vascular progenitors in zebrafish. Genes Dev 1998, 12:621-626. These papers [28••,29••] define a role for SCL in the development of both hematopoietic and vascular development in zebrafish. Gering et al. [28••] show that ectopic expression of SCL in zebrafish embryos results in the overproduction of both hematopoietic and endothelial precursors [28••]. Liao et al. [29••] find that the hematopoietic and angiogenic defects of cloche are corrected by enforced expression of SCL, thus demonstrating that SCL acts downstream of cloche. More work is needed to resolve whether SCL actually functions to specify the formation of a hemangioblast stem cell in mice and zebrafish, or whether it only acts separately in the development of both lineages. 30. Warren AJ, Colledge WH, Carlton MBL, Evans MJ, Smith AJH, Rabbitts TH: The oncogenic cysteine-rich LIM domain protein rbtn2 is essential for erythroid development. Cell 1994, 78:45-58. 31. Yamada Y, Warren AW, Dobson C, Forster A, Pannell R, Rabbitts TH: • The T cell leukemia LIM protein Lmo2 is necessary for adult mouse hematopoieses. Proc Natl Acad Sci USA 1998, 95:3890-3895. See annotation [33•]. Transcription factors in hematopoiesis Engel and Murre 32. Wadman IA, Osada H, Grutz GG, Agulnick AD, Westphal H, Forster A, Rabbitts TH: The LIM-only protein Lmo2 is a bridging molecule assembling an erythroid, DNA-binding complex which includes the TAL1, E47, GATA-1 and Ldb/NL1 proteins. EMBO J 1997, 16:3145-3157. 33. Cohen-Kaminsky S, Maouche-Chretien L, Vitelli L, Vinit M-A, Blanchard I, • Yamamoto M, Peschle C, Romeo P-H: Chromatin immunoselection defines a TAL-1 target gene. EMBO J 1998, 17:5151-5160. Yamada et al. [31•] used an ES/chimera approach to confirm that LMO2 is required for adult hematopoiesis, consistent with previous data suggesting that it functions together with SCL. Wadman et al. [32] and CohenKaminsky et al. [33•] characterized the sequences bound by complexes defined in erythroid cells as containing SCL and LMO2. 34. Bash RO, Hall S, Timmons CF, Crist WM, Amylon M, Smith RG, Baer R: Does activation of the TAL1 gene occur in a majority of patients with T-cell acute lymphoblastic leukemia? A Pediatric Oncology Group study. Blood 1995, 86:666-676. 35. Ono Y, Fukuhara N, Yoshie O: Transcriptional activity of TAL1 in T cell acute lymphoblastic leukemia (T-ALL) requires RBTN1 or -2 and induces TALLA1, a highly specific tumor marker of T-ALL. J Biol Chem 1997, 272:4576-4581. 36. Rabbitts TH: LMO T-cell translocation oncogenes typify genes activated by chromosomal translocations that alter transcription and developmental processes. Genes Dev 1998, 12:2651-2657. 37. • Grutz GG, Bucher K, Lavenir I, Larson T, Larson R, Rabbitts TH: The oncogenic T cell LIM-protein Lmo2 forms part of a DNA-binding complex specifically in immature T cells. EMBO J 1998, 17:4594-4605. See annotation [39•]. 38. Krosl G, He G, Lefrancois M, Charron F, Romeo P-H, Jolicoeur P, •• Kirsch IR, Nemer M, Hoang T: Transcription factor SCL is required for c-kit expression and c-Kit function in hemopoietic cells. J Exp Med 1998, 188:439-450. See annotation [39•] 39. Ono Y, Fukuhara N, Yoshie O: TAL1 and LIM-only proteins • synergistically induce retinaldehyde degydrogenease 2 expression in T-cell acute lymphoblastic leukemia by acting as cofactors for GATA3. Mol Cell Biol 1998, 18:6939-6950. The complexes described by Grutz et al., found in T-lymphomas and thymocytes overexpressing LMO2 [37•], differ from those characterized in [32] and [33•] in that they do not appear to include a GATA family member. However, Ono et al. [39•] describe a DNA-binding site for a SCL/LMO/GATA-3 complex in T-ALLs. This site, which is found to be requires for SCL/LMO-dependent expression of the RALDH2 gene, requires only an intact GATA sequence for activity. Krosl et al. [38••] use antisense and dominant-negative approaches to inhibit SCL in a hematopoietic line and find that the expression of c-kit was SCL-dependent. Krosl et al. also define a potential SCL binding site in the c-kit promoter that is similar to that found by Grutz et al. [37•]. 40. Hsu H-L, Wadman I, Tsan JT, Baer R: Positive and negative transcriptional control by the TAL1 helix-loop-helix protein. Proc Natl Acad Sci USA 1994, 91:5947-5951. 41. Park ST, Sun X-H: The Tal1 oncoprotein inhibits E47-mediated transcription. Mechanism of inhibition. J Biol Chem 1998, 273:7030-7037. 42. Bain G, Engel I, Maandag ECR, te Riele HPJ, Voland JR, Sharp LL, Chun J, Huey B, Pinkel D, Murre C: E2A deficiency leads to abnormalities in αβ T-cell development and to rapid development of T-cell lymphomas. Mol Cell Biol 1997, 17:4782-4791. 43. Yan W, Young AZ, Soares VC, Kelley R, Benezra R, Zhuang Y: High Incidence of T-cell tumors in E2A-null mice and E2A/Id1 doubleknockout mice. Mol Cell Biol 1997, 17:7317-7327. 44. Park ST, Nolan GP, Sun X-H: Growth inhibition and apoptosis due • to restoration of E2A activity in T cell acute lymphoblastic leukemia cells. J Exp Med 1999, 189:501-508. See annotation [45•] 45. Engel I, Murre C: Ectopic expression of E47 or E12 promotes the • death of E2A-deficient lymphomas. Proc Natl Acad Sci USA 1999, 96:996-1001. These papers [44•,45•] suggest that E2A functions a tumor suppressor in the T-cell lineage. Park et al. [44•] enforce in the Jurkat T-ALL line the expression 579 of a chimeric protein consisting of the bHLH domain of SCL and amino-terminal domains of E2A. This protein thus binds DNA only as a heterodimer with endogenous type I bHLH proteins such as those encoded by E2A or HEB (similar to wild-type SCL), but contained the transactivation potential of wild-type E2A. Park et al. find that the E2A–SCL construct induces both apoptosis and inhibits DNA synthesis when retrovirally transduced into Jurkat cells. We have transduced both the E47 and E12 products of the E2A gene into cell lines derived from lymphomas that arose in E2A-deficient mice [45•]. We observe that either E2A-encoded protein act to kill these lines, but unlike Park et al., find no effect on cell-cycle progression. 46. Nichogiannopoulou A, Trevisan M, Friedrich C, Georgopoulos K: Ikaros in hemopoietic lineage determination and homeostasis. Sem Immunol 1998, 10:119-125. 47. •• Wang J-H, Avitahl N, Cariappa A, Friedrich C, Ikeda T, Renold A, Andrikopoulos K, Liang L, Pillai S, Morgan BA, Georgopoulos K: Aiolos regulates B cell activation and maturation to effector state. Immunity 1998, 9:543-553. See annotation [50•]. 48. Brown KE, Guest SS, Smale ST, Hahm K, Merkenschlager M, Fisher AG: Association of transcriptionally silent genes with Ikaros complexes at centromeric heterochromatin. Cell 1997, 91:845-854. 49. Avitahl N, Winandy S, Friedrich C, Jones B, Ge Y, Georgopoulos K: •• Ikaros sets thresholds for T cell activation and regulates chromosome propogation. Immunity 1999, 10:333-343. See annotation [50•]. 50. Kim J, Sif S, Jones B, Jackson A, Koipally J, Heller E, Winandy S, Viel • A, Sawyer A, Ikeda T et al.: Ikaros DNA-binding proteins direct formation of chromatin remodeling complexes in lymphocytes. Immunity 1999, 10:345-355. Reports from the Georgopolous group [47••,49••,50•] suggest new models for how the Ikaros family acts to influence hematopoietic development. Avitahl et al. [49••] find that Ikaros functions to repress T-cell activation induced by a variety of stimuli. As the stimulatory pathways tested are unlikely to converge on the same target genes, Avitahl et al. reason that Ikaros is probably acting as a general repressor of activation, raising the strength of the stimuli needed to generate a response. Avitahl et al. also report that Ikaros proteins are found in toroidal chromatin structures that co-localize with DNA replication complexes, and that cycling T-cells with reduced Ikaros levels rapidly accumulate chromosomal abnormalities. On the basis of the phenotypes of T-cells with normal and reduced Ikaros levels, Avitahl et al. propose [49••] that the functions of Ikaros to set activation thresholds and maintain chromosomal integrity were likely related to its assembly into higher-order chromatin structures. Wang et al. [47••] report that Aiolos-deficient mice exhibit hyperactive B-cell responses, suggesting that the role of Aiolos in B-cells is likely to have many similarities to that of Ikaros in T-cells. Kim et al. [50•] find that both Ikaros and Aiolos are associated with chromatinremodeling and histone deacetylation complexes in T-cells. 51. Geiger H, Sick S, Bonifer C, Muller AM: Globin gene expression is •• reprogrammed in chimeras generated by injecting adult hematopoietic stem cells into mouse blastocysts. Cell 1998, 93:1055-1065. Geiger et al. use tranplantation of embryonic/fetal and adult HSCs into adult and blastocyst host environments, respectively, to examine the influence of microenvironment on the expression of a transgenic human globin locus. They find that both adult and embryonic hosts function in a dominant fashion to reprogram gene expression of the transplanted HSCs. 52. Zhao K, Wang W, Rando OJ, Xue Y, Swiderek K, Kuo A, Crabtree GR: •• Rapid and phosphoinositol-dependent binding of the SWI/SNFlike BAF complex to chromatin after T lymphocyte receptor signaling. Cell 1998, 95:625-636. See annotation [53•]. 53. Agarwal S, Rao A: Modulation of chromatin structure regulates • cytokine gene expression during T cell differentiation. Immunity 1998, 9:765-775. These reports provide evidence for chromatin remodeling upon T-cell activation and differentiation. Zhao et al. [52••] report that phosphatidyl inositol 4,5-bisphosphate (PIP2) generated by treatment of T-cells with anti-TCR antibodies or pharmacological mimics induced the association to chromatin of the BAF complex, which contains subunits related to the yeast SWI/SNF and Drosophila Brahma proteins [52••]. Agarwal and Rao [53•] find that differentiation of naïve T-helper populations into different polarized T-helper subsets (i.e. Th1 and Th2) is associated with changes in chromatin structure at the appropriate gene loci, as determined by Dnase hypersensitivity.