STEM CELL TRANSCRIPTION FACTORS

APLASTIC ANEMIA AND STEM CELL BIOLOGY

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STEM CELL TRANSCRIPTION FACTORS Ramesh A. Shivdasani, MD, PhD

THE PROBLEM WITH STEM CELL BIOCHEMISTRY

The functional definition of the hematopoietic stem cell (HSC) is based on rescue of most or all blood cell lineages in lethally irradiated mice, whereas the phenotypic definition has relied on, among other criteria, immunologic markers and exclusion of fluorochromes. Cell populations that are greatly enriched for the HSC, however, invariably include progenitors already committed to differentiate into one or more daughter lineages. This difficulty in isolating a homogeneous population of "true" stem cells has made it difficult to use biochemical or gene expression studies to arrive at conclusions about the expression and function of individual transcription factors within the earliest blood cell. Morrison et all5have argued persuasively that the difficulties inherent in pinpointing a specific stem cell should not detract from addressing questions of stem cell biology, and they further suggest that stem cells be considered to include all self-renewing progenitor cells with the broadest developmental potential available within a particular tissue at a particular time. Although transplantation, immunologic, and growth factor studies have yielded useful information about the biology of HSCs, they have been less informative about the roles of individual transcription factors. Nevertheless, valuable insights into stem cell transcription factor function have emerged from targeted gene disruption (knockout) studies in mice. Rather than a discussion of all aspects of putative stem cell transcription factors, this article provides a brief summary of how selected gene knockout studies have contributed to the identification of critical transcription factors in the biology of HSCs as we understand them today. It is useful to begin this discussion by considering individual properties of Supported in part by Grant HL 03290 from the National Institutes of Health. From the Dana-Farber Cancer Institute, and Harvard Medical School, Boston, Massachu-

setts HEMATOLOGY/ONCOLOGY CLINICS OF NORTH AMERICA ~

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HSCs that must depend on the functions of specific proteins. Self-renewal, maintenance of the pluripotential state, and the presumed ability to undergo asymmetric cell divisions are stem cell properties that must at some level be regulated by specific transcription factors, yet the identity of the key factors in stem cells of any tissue is currently unknown. As stem cells differentiate, their more mature progeny manifest distinct patterns of gene expression that must be controlled by separate, albeit possibly overlapping, sets of transcription factors. Studies in several model systems are converging toward the notion that tissuespecific gene expression depends on the presence of the right combination of general and lineage-restricted transcription factors in the differentiated cell. Understanding the transition between stem cells and differentiated precursors, particularly as they relate to patterns of gene expression established by individual transcription factors, represents an important challenge in the study of cell differentiation. TAL1, A bHLH TRANSCRIPTION FACTOR THAT FUNCTIONS IN THE EARLIEST STAGES OF HEMATOPOIESIS

The transcription factor TALl (for T-cell acute leukemia-1), also known as SCL (for stem cell leukemia) and TCL5, was originally identified as the product of a gene locus that is occasionally translocated in T-cell acute leukemias (T-ALL).STranslocation near the T-cell receptor S chain gene leads to high-level, ectopic expression within maturing T lymphocytes and presumably contributes to neoplastic transformation. Notably, the leukemic cell line used for the initial cloning could be induced to differentiate into either myeloid or lymphoid cells in vitro and was derived from a patient whose lymphoblasts exhibited myeloid features after treatment with cytotoxic chemotherapy. Accordingly, the product of the unrearranged TALl locus was believed to function in some fundamental aspect of blood cell differentiation. In addition, its primary amino acid sequence predicts a secondary structure that includes an amphipathic helix-loop-helix domain and characteristic, basic dimerization interface, together known as the bHLH motif. Within the latter motif there is strong homology between TALl and several transcription factors known to influence cell fate, including MyoD, related myogenic bHLH proteins, and the daughterless and achaete-scute families of Drosophila proteins.I8 By analogy, it seemed likely that TALl mediated parallel functions in hematopoiesis. Expression of TALl mRNA is restricted to selected cell lineages during embryonic development and in adult tissues. In situ hybridization studies have defined the pattern of expression of this gene to include vascular endothelial cells, neurons in portions of the mesencephalon and metencephalon, and hematopoietic cells.7,lo,11, l6 Among normal hematopoietic cells, TALl transcripts and protein are detected only in erythroid and mast cells and megakaryocytes, whereas studies in immortalized cell lines extend this range of expression to factor-dependent primitive myeloid cells.%Whereas TALl mRNA is absent in most mature myeloid and lymphoid cell lines, it increases during erythroid differentiation in vitro.1,33Inasmuch as expression patterns point to sites of in vivo gene function, these data raise the possibility of distinct TALl functions in early and late hematopoiesis, as well as some aspects of neuronal and vascular function. Two independent gene knockout experiments in mice have established 27 Mouse embryos lacking the the function of TALl in normal development.25* endogenous gene product uniformly die by the 10th day of gestation, when the

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yolk sac is the dominant site of hematopoiesis. These embryos show no evidence of blood formation and presumably die from anemia at the earliest developmental stage in which circulation of red blood cells is essential for tissue respiration. On the other hand, the presence of grossly intact vascular endothelial cells indicates that among cell lineages in which TALl mRNA is present, a severe cellular defect in its absence is restricted to hematopoiesis; early embryonic lethality precludes definition of a clear function in development of the central nervous system. The severe phenotype of TALl knockout mice raises two important questions. First, what is the consequence of TALl loss on definitive (adult) hematopoiesis? Second, does the observed defect solely reflect inactivation of the targeted gene or were additional genes affected inadvertently by recombination in embryonic stem (ES) cells and prolonged cell culture? Cultures of TAL1-I- yolk sacs in a variety of cytokines fail to yield either primitive or definitive hematopoietic cell colonies in vitro.25,27 Furthermore, independent ES cell clones carrying null mutations in both copies of the endogenous TALl gene are incapable of differentiating into any hematopoietic cells in vitro" or in chimeric mice,= a striking defect that is rescued by a single copy of the gene introduced in trans into the TALl - I - ES cells.= The sum of these data proves that TALl is required for development of all primitive (embryonic) as well as definitive (adult) hematopoietic lineages, including lymphocytes. Like other bHLH transcription factors, TALl recognizes an E-box (CANNTG)consensus DNA sequence, although the identity of genes that harbor the TALl consensus sequence CAGATG within relevant cis-elements, and whose expression in immature hematopoietic cells is regulated by TAL1, remains unknown. To bind DNA, TALl dimerizes with the E12/E47 bHLH protein products of the E2A gene locus, and it is for this heterodimer that the CAGATG consensus sequence was defined.8T9The hematopoietic phenotype of E2A knockout mice is largely limited to arrested B-cell maturation? 38 so that its broader requirement in hematopoiesis in vivo appears to be limited. In some cell types, including differentiated myelocytes, the function of bHLH transcription factor complexes is partly regulated at the level of competition for binding to the structurally related Id pr~tein,'~ whose role in HSC or erythroid cell differentiation is still under investigation. LM02, A SMALL NUCLEAR PROTEIN THAT ASSOCIATES WITH TALl AND REGULATES HEMATOPOIESIS

The phenotype of TAL1-null mouse embryos closely resembles that of mice carrying a targeted disruption of the gene variously known as LMO (for Limonly) 2, Rbtn2, and Ttg2.37The small (156 amino acids) LM02 protein is characterized by two cysteine-rich LIM domains that are structurally homologous to the zinc-finger DNA binding domain of the GATA family of transcription factors. Like TAL1, the LM02 gene was also originally identified on the basis of translocation in human T-cell acute lymphocytic leukemia (ALL); however, LM02 appears to have a broader expression pattern in the developing mouse, where its mRNA can be detected in brain rhombomeres, hematopoietic tissu.es, and probably other sites as well. The dominance of the hematopoietic phenotype of LMO-2-null and TAL1-null mice, reflecting a developmental arrest with lethal consequences, does not exclude the possibility that these nuclear proteins are also required for the proper differentiation of other cell types. Although TAL1-Ihomozygous mutant ES cells contribute to all nonhematopoietic tissues tested

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in chimeric 24 this analysis was conducted at the level of gross tissues, leaving open the possibility of subtle derangements affecting selected cell populations within individual tissues. The phenotypes of mice lacking either TALl or LM02 are distinct from those of mice carrying a targeted disruption of any other hematopoietic transcription factor gene, where either the defect is quantitatively less severe or selected blood cell lineages are spared (reviewed in ref. 28). These phenotypes suggest that the TALl and LM02 proteins are necessary to mediate either HSC differentiation from uncommitted mesodermal precursors or some very early aspect of stem cell expansion or differentiation into daughter lineages. (The failure of TAL1-/- hematopoietic tissues to generate mature blood cells in vitro or in vivo is consistent with either possibility.) The extent and nature of the hematopoietic abnormality resulting from their absence thus lies at the heart of studies on stem cell transcription factors. Transcripts encoding other hematopoietic-specific transcription factors (p45 NF-E2, EKLF, and c-Myb) can be detected by reverse transcription-polymerase chain reaction (RT-PCR) in differentiated embryoid bodies from TAL1-/- ES cellsz4but not in TAL1-null yolk sacs.25,27 Therefore, it has been difficult to establish unequivocally whether the most primitive hematopoietic progenitors are ever generated in the absence of TALL Ectopic expression of TALl protein in the leukemic done is seen in the majority of cases of T-ALL, often by mechanisms other than chromosomal translocation or interstitial deletion4; a fraction of these malignant clones also reveal dysregulated LM02 expression? Thus, TALl and LM02 may fulfill parallel roles in normal hematopoiesis as well as in leukemogenesis. Indeed, the two proteins are not only coexpressed in maturing erythroid cells3' but appear to be associated in a physical complex. They interact with each other in yeast twohybrid assays%and can be co-immunoprecipitated from erythroid cell lines,32as well as from T lymphocytes in mice carrying both cDNAs as transgenes under the regulation of T-cell-specific prom~ters.'~ Although the full composition of the relevant multiprotein complex and its cellular functions remain unknown, experimental evidence suggests that one LIM domain of LM02 interacts with part of the bHLH region of TAL1.32,34 LM02 itself does not bind DNA and thus presumably either modifies TALl DNA binding or bridges contact with other components of a larger complex. This physical association not only suggests that TALl and LM02 participate in a common biochemical pathway but also is particularly intriguing in light of emerging concepts about the transcriptional control of cell differentiation. Thus, in one possible scenario, the requirement for TALl in gene regulation in hematopoietic cells may depend on assembly of a multiprotein complex that includes LM02 and probably other nuclear proteins as well. Other functions of TALl in individual blood cell lineages or in neuronal development may depend on distinct multiprotein complexes, albeit with one or more overlapping components. It is probably the precise site and timing of availability of the right complement of accessory proteins that establishes lineage-specific programs of gene expression and drives cell differentiation. At this time, however, the mechanisms by which lineage-restricted transcription factors may influence progressive differentiation of pluripotential stem cells remain obscure. One remarkable aspect of the TAL1-LM02 interaction is that a large proportion of the total cellular pool of each protein is present within the putative multiprotein complex, at least in erythroid cells3z;the existence of similar complexes has not been demonstrated in more primitive progenitors. In contrast, a suggested interaction between LM02 and GATA-family transcription factors might involve at most a small fraction of the cellular pools of each protein and

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is very difficult to revea1.2O Recall also that TALl binds DNA as a heterodimer with the E12/E47 bHLH protein products of the E2A gene locus, adding further complexity to putative multiprotein complexes of stem cell transcription factors. Finally, any association between the latter and proteins that either modify chromatin structure or facilitate transcription by RNA polymerase I1 has not yet been demonstrated. GATA-2, A ZINC-FINGER TRANSCRIPTION FACTOR WITH POSSIBLE FUNCTIONS IN DRIVING HSC EXPANSION

The GATA family of zinc-finger transcription factors has been the subject of much attention with respect to hematopoiesis. GATA-1, the founding member of this family, is essential for differentiation, survival, and regulated proliferation of red blood cells" and megakaryocytes,26whereas the related protein GATA-3 is required for proper fetal liver hematopoiesis and T-lymphocyte development in vivo.21,29 Mouse embryos lacking GATA-Z die of severe anemia during the early gestational phase of yolk sac hematopoiesis; in contrast to TAL1-/- embryos, however, embryonic red blood cells are not totally absent but only decreased in number.30Although most hematopoietic lineages can be cultured from GATA-2-/- yolk sacs, and GATA-2-/- ES cells contribute to adult blood cells in chimeric mice, the multipotential progenitors proliferate poorly and generate small colonies with excess cell death.30,31 Mast cells are notably absent from long-term cultures. One interpretation of these findings is that GATA-2 is not required for HSC differentiation per se but rather for expansion of either the stem cell itself or of all committed progenitors; in addition, it appears to be necessary for mast cell proliferation and differentiation. These results are consistent with studies in avian erythroid precursors, where forced expression of GATA-2 promotes cell proliferation at the expense of differentiation? As with TALl and LM02, neither the precise progenitor/stem cell in which GATA-2 function is essential nor its critical transcriptional targets are known. CBF, A TRANSCRIPTION FACTOR REQUIRED FOR GENERATION OF ALL DEFINITIVE (ADULT) HEMATOPOIETIC CELL LINEAGES

Like TAL1, one of the genes (AML1) encoding the DNA-binding a subunit of core binding factor (CBF, also known as PEBPZ) is frequently translocated in human myeloid and lymphoid leukemias (reviewed in ref. 36). Similarly, the gene encoding the structurally unrelated p subunit of this transcription factor, which does not bind DNA directly but increases the affinity of the a/@ heterodimer for specific DNA sequences, is occasionally targeted by chromosomal rearrangements in myeloid leukemias. Rather than cause dysregulated gene expression directly, however, each of these chromosomal rearrangements results in the creation of a chimeric transcription factor that includes some portion of the a or p subunit of CBF. The parallels with TALl and LMO2 do not end with their respective roles in hematopoietic malignancies. Mice lacking either the AML1-encoded a subunit or the solitary p subunit of CBF reveal a failure of fetal liver (definitive) hematopoiesis in vivo whereas yolk sac (primitive) hematopoiesis is entirely spared.19, 36 This failure of definitive hematopoiesis is manifested either as complete absence (CBFa-I-) or at least 100-fold reduction (CBFp- I - ) of definitive erythroid and myeloid precursors and circulating cells.

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ES cells carrying homozygous mutations in CBFa do not contribute to hematopoiesis in chimeric mice, whereas CBFP-1- ES cells contribute only weakly. Both mutant mouse strains further exhibit perivascular edema, cell death, and hemorrhage in specific regions of the developing nervous system, but the pathophysiologic basis for these findings is unclear. The apparent effects on all definitive hematopoietic cell lineages imply a function for CBF in very primitive hematopoietic progenitors. Candidate genes for transcriptional regulation by CBF include a number of hematopoietic-specific markers, cytokines, and cytokine receptors, but the critical targets responsible for the severe phenotype in the preceding knockout mice is not known. The remarkable feature of the CBFa and CBFp knockout mice is the sparing of embryonic/primitive hematopoiesis, which is reminiscent of the phenotype of c-Myb-/- mice, except that in the latter instance development of the megakaryocyte lineage is curiously unaffe~ted.'~ Together, these in vivo experiments point to fundamental differences between the genetic programs that control primitive versus definitive hematopoiesis, at least at the level of key transcriptional regulators. This is particularly intriguing in view of recent evidence that both primitive and definitive hematopoietic progenitors may derive from a common precursor.lz Thus, HSCs appear to respond to the hematopoietic microenvironments provided by the embryonic yolk sac and fetal liver with distinct transcription factor requirements for critical proliferation and differentiation functions. C0NCLUSI0N The difficulty with defining HSCs for the purpose of biochemistry and gene expression studies has extended to the characterization of transcription factors that regulate critical HSC functions. Nevertheless, gene knockout studies have complemented a host of other experimental approaches to establish a handful of nuclear proteins as key regulators of either the HSC itself or of very primitive multipotential progenitors. Here I have discussed five such proteins whose absence affects all blood cell lineages and argued that they likely function at the level of the most primitive hematopoietic progenitors. Other transcription factors also play important roles in cell differentiation but appear to exercise this importance within a more restricted range of cell types (reviewed in ref. 28). In all cases, transcription factor function is likely to involve multiprotein complexes that include tissue-restricted and widely expressed components, characterization of which will greatly increase our understanding of the molecular control of cell differentiation. Although the studies summarized here point to some key transcriptional regulators of HSC functions, important questions regarding their mechanisms of action remain unanswered. Progress in this area depends in part on improved characterization of the HSC and primitive committed progenitors. Without the ability to isolate or define reliably the latter cell populations, many hypotheses cannot be tested rigorously, and the true functions of stem cell transcription factors may be difficult to establish. Another challenge is identifying the important targets of transcriptional regulation by putative stem cell transcription factors. Without this information, the link between transcription factors and cell differentiation remains an open loop that begs mechanistic closure. Finally, hematopoiesis responds to external cues, including secreted factors and cell-cell interactions; the effects of these signals on expression and function of stem cell and lineage-restricted transcription factors will provide much needed insight into how hematopoiesis is regulated.

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23. Porcher C, Swat W, Rockwell K, et al: The T-cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages. Cell 8647, 1996 24. Robb L, Elwood NJ, Elefanty AG, et al: The scl gene product is required for the generation of all hematopoietic lineages in the adult mouse. EMBO J 15:4123, 1996 25. Robb L, Lyons I, Li R, et al: Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene. Proc Natl Acad Sci USA 92:7075, 1995 26. Shivdasani RA, Fujiwara Y, McDevitt MA, Orkin S H A lineage-selective knockout establishes the critical role of transcription factor GATA-1 in megakaryocyte growth and platelet development. EMBO J 16:3965-3973, 1997 27. Shivdasani RA, Mayer EL, Orkin S H Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1 /SCL. Nature 373:432, 1995 28. Shivdasani RA, Orkin SH: The transcriptional control of hematopoiesis. Blood 874025, 1996 29. Ting C-N, Olson MC, Barton KE', Leiden JM: The GATA-3 transcription factor is required for the development of the T cell lineage. Nature 384:474, 1996 30. Tsai F-Y, Keller G, Kuo FC, et al: An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature 371:221, 1994 31. Tsai F-Y, Orkin S H Transcription factor GATA-2 is required for proliferation/survival of early hematopoietic cells and mast cell formation, but not for erythroid and myeloid terminal differentiation. Blood 89:363&3643, 1997 32. Valge-Archer VE, Osada H, Warren AJ, et a1 The LIM protein RBTNZ and the basic helix-loop-helix protein TALl are present in a complex in erythroid cells. Proc Natl Acad Sci USA 91:8617,1994 33. Visvader J, Begley CG, Adams J M Differential expression of the Lyl, SCL, and E2a helix-loop-helix genes within the hemopoietic system. Oncogene 6187, 1991 34. Wadman I, Li J, Bash RO, et al: Specific in vivo association between the bHLH and LIM proteins implicated in human T cell leukemia. EMBO J 13:4831, 1994 35. Wang Q, Stacy T, Binder M, et al: Disruption of the CBFa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis. Proc Natl Acad Sci USA 933444,1996 36. Wang Q, Stacy T, Miller JD, et al: The CBFP subunit is essential for CBFa2 (AML1) function in vivo. Cell 87697, 1996 37. Warren AJ, Colledge WH, Carlton MBL, et a1 The oncogenic cysteine-rich LIM domain protein Rbtn2 is essential for erythroid development. Cell 7845, 1994 38. Zhuang Y, Soriano P, Weintraub H The helix-loop-helix gene E2A is required for B cell formation. Cell 792375, 1994

Address reprint requests to Ramesh A. Shivdasani, MD, PhD Dana-Farber Cancer Institute 44 B m e y Street Boston, MA 02115