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Ikaros in hemopoietic lineage determination and homeostasis
seminars in I M M U N OL OG Y, Vol 10, 1998: pp. 119]125
Ikaros in hemopoietic lineage determination and homeostasis Aliki Nichogiannopoulou, Maryanne Trevisan, Christof Friedrich and Katia Georgopoulos the embryo proper ŽT. Ikeda, in preparation.. Ikaros is subsequently seen in the fetal liver primordium from the onset of its development as a hemopoietic tissue Žday 9.5. through mid-late gestation.1 In the developing fetal thymus Ikaros expression is detected from the time of the first seeding by lymphoid precursors Žday 11.5.. Within the hemopoietic system, Ikaros message is expressed at its highest levels within the immature fetal and adult thymocytes. Mature T and B cells as well as natural killer ŽNK. cells and antigen presenting cells ŽAPC. express Ikaros but at lower levels. Low level Ikaros expression is also found in populations enriched for erythroid and myeloid precursors, but is undetectable upon terminal differentiation to erythrocytes, monocytes and macrophages with the exception of mature granulocytes.1,3,4 Ikaros is also expressed in hemopoietic populations which are highly enriched for stem cell activity ŽLiny c-kit q Sca-1 q ..3 ] 6 The pre- and early hemopoietic pattern of Ikaros expression suggests it may regulate cell fate decisions during hemopoietic development.
Studies on the molecular mechanisms that control hemopoietic differentiation have focused on signaling cascades and nuclear effectors that drive this complex developmental system in a regulated fashion. Here we review the role of Ikaros, the founding member of a unique family of zinc finger transcription factors in this developmental process. Studies on an Ikaros null mutation have revealed an essential role for this factor in lymphoid cell fate determination and at subsequent branch points of the T cell differentiation pathway. Differences in the phenotypes of a null and a dominant negative (DN) Ikaros mutation provide insight into a regulatory network through which Ikaros proteins exert their effects in development. In addition a comparative analysis of the hemopoietic stem cell and precursor compartment resulting from the two Ikaros mutations reveals a profound yet not absolute requirement for Ikaros in the production and differentiation of these populations. Key words: hemopoiesis r Ikaros r lymphocyte development Q1998 Academic Press Ltd
Molecular and biochemical properties
Ikaros expression in the hemopoietic system
Ikaros encodes, by means of alternative splicing of exons 3]6, a family of six zinc finger containing proteins which are expressed in the hemopoietic system of mice and humans.1,2,7,8 These proteins contain up to six zinc fingers which are organized into two functionally distinct domains. The N-terminus of Ikaros isoforms contains from zero to four zinc finger motifs which dictate sequence specificity and DNA affinity ŽFigure 1.. Of the six Ikaros isoforms only three, Ik-1, Ik-2 and Ik-3, possess an appropriate N-terminal zinc finger domain capable of high affinity DNA-binding to sites that contain the GGGA base pair core motif ŽFigure 1..8 In contrast to the differential usage of the N-terminal zinc finger motifs, the two C-terminal zinc finger motifs responsible for
IKAROS WAS ORIGINALLY identified as a potential regulator of enhancer and promoter elements critical for the expression of lymphoid-specific genes.1,2 However, its expression is more widely distributed within the developing and adult hemopoietic system. During mouse embryonic development, Ikaros mRNA is first detected in the day 8 blood islands of the yolk sac and in a small number of mesodermal cells within
From The Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA Q1998 Academic Press Ltd 1044-5323r 98r 020119q 07 $25.00r 0r si980113
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mediating protein]protein interactions are present in all Ikaros isoforms. Additionally Ikaros proteins share at least one transcriptional activation domain located within exon 7 Žsee Ref 9 and J. Koipally unpublished results.. Formation of homo- and heterodimers between DNA-binding isoforms ŽIk-1, Ik-2 and Ik-3 . increases their DNA affinity and allows transcriptional activation from their cognate sites.9 However, heterodimers of isoforms with and without DNA-binding domains cannot bind DNA and are transcriptionally inert.9 As a result, Ikaros isoforms lacking a functional DNA-binding domain can interfere with the activity of other isoforms through a dominant negative mechanism. Ik-1 and Ik-2 are the predominant isoforms while Ik-3, Ik-4, Ik-5 and Ik-6 are present at significantly lower levels in all hemopoietic cell populations studied ŽFigure 1..3
includes the C-terminal dimerization zinc fingers and the transcriptional activation domain, was deleted Ž D 7..10 Ikaros proteins generated by the mutant allele are unstable and virtually undetectable; hence mice homozygous for this mutation are considered null for Ikaros activity. In the second mutation exons 3 and 4, which encode a major part of the DNA-binding domain of Ikaros proteins, were deleted Ž D 3]4..11 Proteins lacking a DNA-binding domain but which have intact zinc finger dimerization motifs are stably produced from this mutant allele. These mutant Ikaros proteins are structurally related to the naturally occurring short Ikaros isoforms normally expressed at low levels in the hemopoietic system ŽFigure 1.. Thus, Ikaros isoforms capable of functioning as dominant negatives are produced in excess in the hemopoietic system of mice heterozygous and homozygous for the Ikaros D 3]4 deletion.
Targeting Ikaros mutations in the mouse germ line
Severe defects in lymphocyte development in the absence of Ikaros
To determine the role of Ikaros in hemopoiesis, the Ikaros locus was targeted by two distinct mutations. In the first mutation the coding region of exon 7, which
Ikaros null homozygous mice display a severe defect in lymphopoiesis, manifested in part as an early and
Figure 1. Schematic representation of Ikaros isoforms. Zinc finger domains involved in DNAbinding and dimerization are indicated by horizontal arrows. Asterisks indicate isoforms produced from the DN allele. Ik-6 is also produced from the wild type locus.
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differentiation into single positive CD4q and CD8q ab T cells. However, CD44qHSAqCD25qCD4y CD8y TCRy thymocyte precursors are severely Ž10]20-fold . reduced in number in both young and adult Ikaros null mice, suggesting either a decrease in the input of bone marrow derived lymphoid progenitors or a failure in the expansion of the earliest thymocyte precursors ŽCD4 lo CD44q HSAy ., which is IL-7 and kit ligand dependent and may require Ikaros ŽJ.-H. Wang and K. Georgopoulos, unpublished results.. Although Ikaros null thymocyte precursors progress along the ab T cell lineage, they display a preferred commitment to the CD4q cell fate.10 This suggests that Ikaros may control differentiation at the CD4qrCD8q branch point, possibly by negatively regulating the CD4 gene. Mature CD4q and CD8q ab T cells produced in Ikaros null mice display a hyperproliferative response to TCR stimulation in vitro, a finding consistent with the observed clonal
complete block in B cell differentiation.10 Conventional B cells and the fetally derived B-1a cells are absent from the spleen, the peritoneal cavity and the fetal liver of these mice. In addition, pre-B and pro-B cell precursors are absent from the bone marrow and fetal liver of these mutants. Therefore in the absence of Ikaros there appears to be a lack of B cell specification from either hemopoietic stem cells or a putative common lymphoid precursor ŽFigure 2..12 Loss of Ikaros activity also has severe effects on the differentiation of the fetal and adult T cell lineages. Throughout fetal development and for the first few days after birth, the thymus of Ikaros null homozygous mice is devoid of all identifiable lymphoid precursors.10 Consistent with this lack of fetal T cell differentiation, the fetally derived epidermal gd T cells Žwith Vg 3 specificity. are absent from the skin of Ikaros null mice.10 Between days 3 and 5 postpartum, a small number of T cell precursors are detected in the thymus and subsequently undergo expansion and
Figure 2. Effects of Ikaros mutations in the hemopoietic system. A hierarchical model of hemopoiesis depicting a select number of developmental stages affected by two distinct Ikaros mutations is shown. A role for Ikaros in stem cell function is revealed by the Ikaros null Ž Ik . and dominant negative ŽDN. mutations. Blocks in lymphoid differentiation ŽX. and loss of cell cycle control Žcurved arrows. in the T cell lineage result from these mutations. Loss of Ikaros selectively blocks fetal ŽX U . but not adult T cell development.
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leukemias and lymphomas with 100% penetrance.21 An invariant feature of these T cell malignancies is the loss of heterozygosity at the Ikaros locus, a common mutation in tumor suppressor gene loci.22 ] 24 Null heterozygotes develop T cell malignancies at a much lower frequency than that observed for DN heterozygotes. The rapid development of T cell malignancies in mice heterozygous for the Ikaros DN mutation suggests DN isoforms interfere with either wild type isoforms andror other related factors. Studies on T cells with normal and reduced levels of Ikaros activity reveal an Ikaros-based threshold that controls T cell activation and cell cycle progression.25 When the levels of Ikaros DNA-binding complexes are reduced, as is the case in Ikaros mutants, fewer TCR signaling events are sufficient to drive quiescent T cells into cycle. Given that Ikaros provides an activation threshold for mature T cells, it is intriguing that its nuclear pattern of staining changes after TCR engagement from a mesh of fine speckles and discrete foci to a network of toroids. Ikaros toroids, which form in G1 and are maintained through S phase, associate with DNA replication clusters in heterochromatic regions. The presence of Ikaros in these heterochromatin-associated structures may be critical for the proper control of DNA replication but may also provide the potential for silenced genes present within these heterochromatic domains to become activated.25,26 Mice homozygous for the Ikaros DN mutation also display a more severe block in lymphocyte development compared to Ikaros null mice. Ikaros DN y ry mice display an early and complete block in the development of all fetal and adult lymphoid lineages ŽFigure 2..11 B and T lymphocytes, NK cells as well as thymic and splenic dendritic APCs are absent in these mice, as are their earliest identifiable progenitors. The more severe lymphoid defects manifest in the Ikaros DN y ry mice compared to the Ikaros null mice reflect the combined effect of loss of Ikaros activity and negative interference by Ikaros mutant isoforms towards related factors present in early hemopoietic precursors.
expansion of T cells in the thymus and in the periphery of mutant mice. NK cells, gd T cells and thymic dendritic APCs are absent or significantly reduced in Ikaros null mice,10,13 suggesting that Ikaros may regulate lineage decisions at the level of the common precursor for these lineages.14 ] 16 Among gd T cells, the extrathymically derived intestinal intraepithelial gd T cells and the adult derived gd T cells present in the thymus and the spleen are either absent or drastically reduced.10 In contrast the mucosal epithelial Vg 4 T cells are found in normal distribution and density. Lineage commitment along the ab or gd T cell lineages occurs in early CD4yCD8yHSAqCD25q thymocyte precursors and may rely on the transcriptional activity of the enhancer elements within the d and a loci which drive the expression of sterile transcripts and promote their recombination.17 ] 19 Ikaros may be required for the activity of the d enhancer thereby providing a positive signal for differentiation along the gd T cell lineage. Lymph node development is also impaired in the absence of Ikaros. Ikaros null mice lack inguinal, axial, cervical and mesenteric lymph nodes, Peyer’s patches and lymphoid follicles in the gastrointestinal tract. Transplantation of wild type bone marrow into these mice repopulates the lymphoid compartment but fails to generate lymph node structures suggesting Ikaros is a critical factor in the temporally regulated formation of peripheral lymphatic centers during development ŽA. Nichogiannopoulou and K. Georgopoulos, unpublished results..
More severe effects on lymphoid differentiation are caused by an Ikaros dominant negative mutation Young Ikaros DN q ry mice appear to develop a normal lymphoid compartment. However, thymocytes and splenic T cells display an augmented proliferative response upon TCR stimulation. In addition, between 2 and 3 months of age thymocytes are highly enriched for CD4 q CD8 int TCR q and CD4 int CD8qTCRq cells, the transitional stages of differentiation during which thymic selection occurs. Moreover, clonal expansions of thymocytes with forbidden TCR specificities20 are frequently observed in these mutants. Together these findings suggest a deregulation in the outcome of negative selection during thymocyte differentiation. Three to sixmonth-old Ikaros DN q ry mice develop T cell
The role of Ikaros in hemopoietic precursors The presence of Ikaros mRNA at the earliest sites of hemopoiesis during ontogeny Žsee Ref 11 and T. Ikeda, in preparation. and in populations enriched for the earliest identifiable hemopoietic precursors3,4 prompted a more detailed analysis of the hemopoietic 122
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genitors ŽLiny c-kitq Sca1q Sca2q . express Helios. Pro-B cells have a low level of Helios expression which is further down-regulated in pre-B and mature B cells. Nevertheless, Helios expression persists in thymic and splenic T cell subpopulations. However, unlike Ikaros, Helios is not expressed in erythroid and myeloid lineages. Hemopoietic expression of Dedalos, the fourth family member, is restricted to bone marrow populations enriched for stem cells and early T cell precursors. These expression data hint at the possible involvement of Helios and Dedalos gene products in the early precursor defects observed in Ikaros DN mutants.
precursor compartment in both Ikaros mutants. Ikaros null bone marrow cellularity is reduced two fold, while splenic cellularity approximates normal levels in spite of the lack of B and T cells. Bone marrow is selectively depleted for immature ŽBFU-E . and mature ŽCFU-E . erythroid precursors but contains near normal numbers of granulocytic, monocytic ŽCFC-GrM . and multipotential ŽCFCmulti. precursors. Loss of Ikaros also reduces the number of spleen colony forming cells ŽCFU-S 14 ..27 Nevertheless Ikaros null bone marrow can radioprotect lethally irradiated recipients.28 Null bone marrow contains cells bearing the hemopoietic stem cell phenotype ŽLiny c-kit q Sca1q . 5,6 at normal frequencies and is able to provide long-term repopulation of erythroid and myeloid lineages in an irradiated host. Yet when co-injected with wild type bone marrow in a competitive repopulation assay,29,30 Ikaros null bone marrow cells fail to contribute to the hemopoietic compartment of the recipient. This latter finding suggests Ikaros null stem cells are either reduced in number or unable to compete effectively with wild type cells in the repopulation of recipients. These data strongly suggest an important yet not essential role for Ikaros in the engraftment, production or maintenance of hemopoietic stem cells or their immediate progeny ŽFigure 2.. The more severe defects manifested in the hemopoietic stem cell compartment of Ikaros DN homozygotes also compel a role for other factors which work in concert with Ikaros and against which DN Ikaros isoforms exert their effects in these processes ŽA. Nichogiannopoulou, in preparation..
Future directions Mice mutant for each of the Ikaros family members are currently being generated and studied to delineate the role of each gene in hemopoietic development. Distinct phenotypes resulting from each mutation will reveal essential and non-overlapping functions for Ikaros, Aiolos, Dedalos and Helios proteins. The presence or absence of novel phenotypes following genetic crossing of single mutants should reveal the degree of functional redundancy between these factors in hemopoietic lineage specification and homeostasis. The molecular mechanisms by which Ikaros exerts its function may relate in part to the transcriptional regulation of genes which are essential for lineage commitment and subsequent differentiation. Differential mRNA analysis between wild type and mutant Ikaros cell populations may identify genetic targets which when inappropriately expressed lead to the profound lymphoid phenotypes observed in Ikaros mutant mice. In mature lymphocytes, Ikaros provides an activation threshold which is overcome by TCR ligation. Changes in Ikaros-containing nuclear structures upon TCR engagement suggest a possible change in Ikaros function from regulating transcription to regulating DNA metabolism during cell cycle. The mechanisms by which Ikaros protein is targeted by TCR signaling cascades and through which Ikaros regulates stable propagation of the genome are currently being investigated. The mesodermal expression pattern of Ikaros is also being exploited to study the earliest events in embryonic and fetal hemopoiesis. By targeting the Lac Z reporter gene into the endogenous Ikaros locus we have a novel strategy for isolating the earliest hemopoietic populations and assessing their de-
Ikaros dimerization partners in the hemolymphoid system. Aiolos, Helios and Dedalos were identified as three novel Ikaros homologues which homo- and heterodimerize with Ikaros and each other ŽSee 3,31 and B.A. Morgan, unpublished results.. Aiolos and Helios proteins show extensive homology to Ikaros DNA-binding, dimerization and activation domains. Aiolos expression is restricted to the lymphoid lineages.3 During embryogenesis, Helios expression pattern resembles that of Ikaros. Helios is detected in day 8.5 yolk sac blood islands and day 9.5 fetal liver although at lower levels than those observed for Ikaros. Its expression in the fetal thymus is not detected until day 15, which is later than Ikaros detection. In the adult bone marrow populations enriched for stem cells ŽLiny c-kit q Sca1q . and lymphoid pro123
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10. Wang J, Nichogiannopoulou A, Wu L, Sun L, Sharpe A, Bigby M, Georgopoulos K Ž1996. Selective defects in the development of the fetal and adult lymphoid system in mice with an Ikaros null mutation. Immunity 5:537]549 11. Georgopoulos K, Bigby M, Wang J-H, Molnar A, Wu P, Winandy S, Sharpe A Ž1994. The Ikaros gene is required for the development of all lymphoid lineages. Cell 79:143]156 12. Kondo M, Weissman IL, Akashi K Ž1997. Identification of clonogenic common progenitors in mouse bone marrow. Cell 91:661]672 13. Wu L, Nichogiannopoulou A, Shortman K, Georgopoulos K Ž1997. Cell-autonomous defects in dendritic cell populations of Ikaros mutant mice point to a developmental relationship with the lymphoid lineage. Immunity 7:483]492 14. Ardavin C, Wu L, Li C, Shortman K Ž1993. Thymic dendritic cells and T cells develop simultaneously in the thymus from a common precursor population. Nature 362:761]763 15. Shortman K, Wu L Ž1996. Early T lymphocyte progenitors. Annu Rev Immunol 14:29]47 16. Wu L, Vremec D, Ardavin K, Winkel G, Suess IL, Maraskovsky E, Cook W, Shortman K Ž1995. Mouse thymus dendritic cells: kinetics of development and changes in surface markers during maturation. Eur J Immunol 25:418]425 17. Taccioli GE, Rathbun G, Shinkai Y, Oltz EM, Cheng H, Whitmore G, Stamato T, Jeggo P, Alt FW Ž1992. Activities involved in VŽD.J recombination. Curr Top Microbiol Immunol 182:107]114 18. Grosschedl R Ž1995. Higher-order nucleoprotein complexes in transcription: analogies with site-specific recombination ŽReview.. Curr Opin Cell Biol 7:362]370 19. Sleckman BP, Bardon CG, Ferrini R, Davidson L, Alt FW Ž1997. Function of the TCR alpha enhancer in alpha beta and gamma delta T cells. Immunity 7:505]515 20. Scherer MT, Ignatowicz L, Winslow GM, Kappler JW, Marrack P Ž1993. Superantigens: bacterial and viral proteins that manipulate the immune system. Annu Rev Cell Biol 9:101]128 21. Winandy S, Wu P, Georgopoulos K Ž1995. A dominant mutation in the Ikaros gene leads to rapid development of leukemia and lymphoma. Cell 83:289]299 22. Cavenee WK, Dryja TP, Phillips RA, Benedict WF, Godbout R, Gallie BL, Murphree AL, Strong LC, White RL Ž1983. Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature 305:779]784 23. Meltzer S, Yin J, Huang Y, McDaniel T, Newkirk C, Iseri O, Vogelstein B, Resau J Ž1991. Reduction to homozygosity involving p53 in esophageal cancers demonstrated by the polymerase chain reaction. Proc Natl Acad Sci USA 88:4976]4980 24. Ichii S, Horii A, Nakatsuru S, Furuyama J, Utsunomiya J, Nakamura Y Ž1992. Inactivation of both APC alleles in an early stage of colon adenomas in a patient with familial adenomatous polyposis ŽFAP.. Hum Mol Genet 1:387]390 25. Avitahl N, Friedrich C, Winandy S, Jones B, Ge Y, Georgopoulos K Ž1998. A macromolecular complex containing Ikaros controls T cell activation and chromosome homeostasis. Žsubmitted for publication. 26. Brown KE, Guest SS, Smale ST, Hahm K, Merkenschlager M, Fisher AG Ž1997. Association of transcriptionally silent genes with Ikaros complexes at centromeric heterochromatin. Cell 91:845]854 27. Till JE, McCulloch EA Ž1961. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res 14:213]222 28. Mulder AH, Visser JW Ž1987. Separation and functional analysis of bone marrow cells separated by rhodamine-123 fluorescence. Exp Hematol 15:99]104
velopmental potential and molecular composition. When combined with tissue explant experiments, this approach will ultimately seek to identify factors that induce Ikaros expression, an event which in the early embryo may be concomitant with mesoderm commitment to hemopoietic cell fate.
Acknowledgements We wish to thank T. Ikeda, J. Koipally, C. Kelley, N. Avitahl, S. Winandy and B. Morgan for constant valuable discussion and the communication of unpublished results. K. Georgopoulos is a Scholar of the Leukemia Society of America. M. Trevisan is an NCIC Terry Fox Junior Research Fellow supported with funds provided by the Terry Fox Run. The transgenic and other research were supported by an ACS grant to K. Georgopoulos and by a core grant from the Cutaneous Biology Research Center ŽShiseido Co. Ltd..
References 1. Georgopoulos K, Moore D, Derfler B Ž1992. Ikaros, an early lymphoid restricted transcription factor and a putative mediator for T cell commitment. Science 258:808]812 2. Hahm K, Ernst P, Lo K, Kim GS, Turck C, Smale ST Ž1994. The lymphoid transcription factor LyF-1 is encoded by specific, alternatively spliced mRNAs derived from the Ikaros gene. Mol Cell Biol 14:7111]7123 3. Morgan B, Sun L, Avitahl N, Andrikopoulos K, Gonzales E, Ikeda T, Wu P, Neben S, Georgopoulos K Ž1997. Aiolos, a lymphoid restricted transcription factor that interacts with Ikaros to regulate lymphocyte differentiation. EMBO J 16:2004]2013 4. Klug AC, Morrison SJ, Masek M, Hahm K, Smale ST, Weisman IL Ž1998. Hematopoietic stem cells and lymphoid progenitors express different Ikaros isoforms, and Ikaros is localized to heterochromatin in immature lymphocytes. Proc Natl Acad Sci USA 95:657]662 5. Okada S, Nakauchi H, Nagayoshi K, Nishikawa S, Nishikawa S-I, Miura Y, Suda T Ž1991. Enrichment and characterization of murine hematopoietic stem cells that express the c-kit molecule. Blood 78:1706 6. Spangrude G, Heimfeld S, Weissman I Ž1988. Purification and characterization of mouse hematopoietic stem cells. Science 241:58]92 7. Molnar A, Wu P, Largespada D, Vortkamp A, Scherer S, Copeland N, Jenkins N, Bruns G, Georgopoulos K Ž1996. The Ikaros gene encodes a family of lymphocyte restricted zinc finger DNA binding proteins, highly conserved in human and mouse. J Immunol 156:585]592 8. Molnar A, Georgopoulos K Ž1994. The Ikaros gene encodes a family of functionally diverse zinc finger DNA-binding proteins. Mol Cell Biol 14:8292]8303 9. Sun L, Liu A, Georgopoulos K Ž1996. Zinc finger-mediated protein interactions modulate Ikaros activity, a molecular control of lymphocyte development. EMBO J 15:5358]5369
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29. Harrison DE Ž1980. Competitive repopulation: a new assay for long-term stem cell functional capacity. Blood 55:77]81 30. Szilvassy SJ, Humphries RK, Lansdorp PM, Eaves AC Ž1990. Quantitative assay for totipotent reconstituting hematopoetic stem cells by a competitive repopulation strategy. Proc Natl Acad Sci USA 87:8736]8740
31. Kelley CM, Ikeda T, Koipally J, Avitahl N, Wu L, Georgopoulos K, Morgan B Ž1998. Helios, a novel dimerization partner of Ikaros, expressed in the earliest hematopoietic progenitors. Curv Biol 8:1]9
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Ikaros in hemopoietic lineage determination and homeostasis Aliki Nichogiannopoulou, Maryanne Trevisan, Christof Friedrich and Katia Georgopoulos the embryo proper ŽT. Ikeda, in preparation.. Ikaros is subsequently seen in the fetal liver primordium from the onset of its development as a hemopoietic tissue Žday 9.5. through mid-late gestation.1 In the developing fetal thymus Ikaros expression is detected from the time of the first seeding by lymphoid precursors Žday 11.5.. Within the hemopoietic system, Ikaros message is expressed at its highest levels within the immature fetal and adult thymocytes. Mature T and B cells as well as natural killer ŽNK. cells and antigen presenting cells ŽAPC. express Ikaros but at lower levels. Low level Ikaros expression is also found in populations enriched for erythroid and myeloid precursors, but is undetectable upon terminal differentiation to erythrocytes, monocytes and macrophages with the exception of mature granulocytes.1,3,4 Ikaros is also expressed in hemopoietic populations which are highly enriched for stem cell activity ŽLiny c-kit q Sca-1 q ..3 ] 6 The pre- and early hemopoietic pattern of Ikaros expression suggests it may regulate cell fate decisions during hemopoietic development.
Studies on the molecular mechanisms that control hemopoietic differentiation have focused on signaling cascades and nuclear effectors that drive this complex developmental system in a regulated fashion. Here we review the role of Ikaros, the founding member of a unique family of zinc finger transcription factors in this developmental process. Studies on an Ikaros null mutation have revealed an essential role for this factor in lymphoid cell fate determination and at subsequent branch points of the T cell differentiation pathway. Differences in the phenotypes of a null and a dominant negative (DN) Ikaros mutation provide insight into a regulatory network through which Ikaros proteins exert their effects in development. In addition a comparative analysis of the hemopoietic stem cell and precursor compartment resulting from the two Ikaros mutations reveals a profound yet not absolute requirement for Ikaros in the production and differentiation of these populations. Key words: hemopoiesis r Ikaros r lymphocyte development Q1998 Academic Press Ltd
Molecular and biochemical properties
Ikaros expression in the hemopoietic system
Ikaros encodes, by means of alternative splicing of exons 3]6, a family of six zinc finger containing proteins which are expressed in the hemopoietic system of mice and humans.1,2,7,8 These proteins contain up to six zinc fingers which are organized into two functionally distinct domains. The N-terminus of Ikaros isoforms contains from zero to four zinc finger motifs which dictate sequence specificity and DNA affinity ŽFigure 1.. Of the six Ikaros isoforms only three, Ik-1, Ik-2 and Ik-3, possess an appropriate N-terminal zinc finger domain capable of high affinity DNA-binding to sites that contain the GGGA base pair core motif ŽFigure 1..8 In contrast to the differential usage of the N-terminal zinc finger motifs, the two C-terminal zinc finger motifs responsible for
IKAROS WAS ORIGINALLY identified as a potential regulator of enhancer and promoter elements critical for the expression of lymphoid-specific genes.1,2 However, its expression is more widely distributed within the developing and adult hemopoietic system. During mouse embryonic development, Ikaros mRNA is first detected in the day 8 blood islands of the yolk sac and in a small number of mesodermal cells within
From The Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA Q1998 Academic Press Ltd 1044-5323r 98r 020119q 07 $25.00r 0r si980113
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mediating protein]protein interactions are present in all Ikaros isoforms. Additionally Ikaros proteins share at least one transcriptional activation domain located within exon 7 Žsee Ref 9 and J. Koipally unpublished results.. Formation of homo- and heterodimers between DNA-binding isoforms ŽIk-1, Ik-2 and Ik-3 . increases their DNA affinity and allows transcriptional activation from their cognate sites.9 However, heterodimers of isoforms with and without DNA-binding domains cannot bind DNA and are transcriptionally inert.9 As a result, Ikaros isoforms lacking a functional DNA-binding domain can interfere with the activity of other isoforms through a dominant negative mechanism. Ik-1 and Ik-2 are the predominant isoforms while Ik-3, Ik-4, Ik-5 and Ik-6 are present at significantly lower levels in all hemopoietic cell populations studied ŽFigure 1..3
includes the C-terminal dimerization zinc fingers and the transcriptional activation domain, was deleted Ž D 7..10 Ikaros proteins generated by the mutant allele are unstable and virtually undetectable; hence mice homozygous for this mutation are considered null for Ikaros activity. In the second mutation exons 3 and 4, which encode a major part of the DNA-binding domain of Ikaros proteins, were deleted Ž D 3]4..11 Proteins lacking a DNA-binding domain but which have intact zinc finger dimerization motifs are stably produced from this mutant allele. These mutant Ikaros proteins are structurally related to the naturally occurring short Ikaros isoforms normally expressed at low levels in the hemopoietic system ŽFigure 1.. Thus, Ikaros isoforms capable of functioning as dominant negatives are produced in excess in the hemopoietic system of mice heterozygous and homozygous for the Ikaros D 3]4 deletion.
Targeting Ikaros mutations in the mouse germ line
Severe defects in lymphocyte development in the absence of Ikaros
To determine the role of Ikaros in hemopoiesis, the Ikaros locus was targeted by two distinct mutations. In the first mutation the coding region of exon 7, which
Ikaros null homozygous mice display a severe defect in lymphopoiesis, manifested in part as an early and
Figure 1. Schematic representation of Ikaros isoforms. Zinc finger domains involved in DNAbinding and dimerization are indicated by horizontal arrows. Asterisks indicate isoforms produced from the DN allele. Ik-6 is also produced from the wild type locus.
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differentiation into single positive CD4q and CD8q ab T cells. However, CD44qHSAqCD25qCD4y CD8y TCRy thymocyte precursors are severely Ž10]20-fold . reduced in number in both young and adult Ikaros null mice, suggesting either a decrease in the input of bone marrow derived lymphoid progenitors or a failure in the expansion of the earliest thymocyte precursors ŽCD4 lo CD44q HSAy ., which is IL-7 and kit ligand dependent and may require Ikaros ŽJ.-H. Wang and K. Georgopoulos, unpublished results.. Although Ikaros null thymocyte precursors progress along the ab T cell lineage, they display a preferred commitment to the CD4q cell fate.10 This suggests that Ikaros may control differentiation at the CD4qrCD8q branch point, possibly by negatively regulating the CD4 gene. Mature CD4q and CD8q ab T cells produced in Ikaros null mice display a hyperproliferative response to TCR stimulation in vitro, a finding consistent with the observed clonal
complete block in B cell differentiation.10 Conventional B cells and the fetally derived B-1a cells are absent from the spleen, the peritoneal cavity and the fetal liver of these mice. In addition, pre-B and pro-B cell precursors are absent from the bone marrow and fetal liver of these mutants. Therefore in the absence of Ikaros there appears to be a lack of B cell specification from either hemopoietic stem cells or a putative common lymphoid precursor ŽFigure 2..12 Loss of Ikaros activity also has severe effects on the differentiation of the fetal and adult T cell lineages. Throughout fetal development and for the first few days after birth, the thymus of Ikaros null homozygous mice is devoid of all identifiable lymphoid precursors.10 Consistent with this lack of fetal T cell differentiation, the fetally derived epidermal gd T cells Žwith Vg 3 specificity. are absent from the skin of Ikaros null mice.10 Between days 3 and 5 postpartum, a small number of T cell precursors are detected in the thymus and subsequently undergo expansion and
Figure 2. Effects of Ikaros mutations in the hemopoietic system. A hierarchical model of hemopoiesis depicting a select number of developmental stages affected by two distinct Ikaros mutations is shown. A role for Ikaros in stem cell function is revealed by the Ikaros null Ž Ik . and dominant negative ŽDN. mutations. Blocks in lymphoid differentiation ŽX. and loss of cell cycle control Žcurved arrows. in the T cell lineage result from these mutations. Loss of Ikaros selectively blocks fetal ŽX U . but not adult T cell development.
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leukemias and lymphomas with 100% penetrance.21 An invariant feature of these T cell malignancies is the loss of heterozygosity at the Ikaros locus, a common mutation in tumor suppressor gene loci.22 ] 24 Null heterozygotes develop T cell malignancies at a much lower frequency than that observed for DN heterozygotes. The rapid development of T cell malignancies in mice heterozygous for the Ikaros DN mutation suggests DN isoforms interfere with either wild type isoforms andror other related factors. Studies on T cells with normal and reduced levels of Ikaros activity reveal an Ikaros-based threshold that controls T cell activation and cell cycle progression.25 When the levels of Ikaros DNA-binding complexes are reduced, as is the case in Ikaros mutants, fewer TCR signaling events are sufficient to drive quiescent T cells into cycle. Given that Ikaros provides an activation threshold for mature T cells, it is intriguing that its nuclear pattern of staining changes after TCR engagement from a mesh of fine speckles and discrete foci to a network of toroids. Ikaros toroids, which form in G1 and are maintained through S phase, associate with DNA replication clusters in heterochromatic regions. The presence of Ikaros in these heterochromatin-associated structures may be critical for the proper control of DNA replication but may also provide the potential for silenced genes present within these heterochromatic domains to become activated.25,26 Mice homozygous for the Ikaros DN mutation also display a more severe block in lymphocyte development compared to Ikaros null mice. Ikaros DN y ry mice display an early and complete block in the development of all fetal and adult lymphoid lineages ŽFigure 2..11 B and T lymphocytes, NK cells as well as thymic and splenic dendritic APCs are absent in these mice, as are their earliest identifiable progenitors. The more severe lymphoid defects manifest in the Ikaros DN y ry mice compared to the Ikaros null mice reflect the combined effect of loss of Ikaros activity and negative interference by Ikaros mutant isoforms towards related factors present in early hemopoietic precursors.
expansion of T cells in the thymus and in the periphery of mutant mice. NK cells, gd T cells and thymic dendritic APCs are absent or significantly reduced in Ikaros null mice,10,13 suggesting that Ikaros may regulate lineage decisions at the level of the common precursor for these lineages.14 ] 16 Among gd T cells, the extrathymically derived intestinal intraepithelial gd T cells and the adult derived gd T cells present in the thymus and the spleen are either absent or drastically reduced.10 In contrast the mucosal epithelial Vg 4 T cells are found in normal distribution and density. Lineage commitment along the ab or gd T cell lineages occurs in early CD4yCD8yHSAqCD25q thymocyte precursors and may rely on the transcriptional activity of the enhancer elements within the d and a loci which drive the expression of sterile transcripts and promote their recombination.17 ] 19 Ikaros may be required for the activity of the d enhancer thereby providing a positive signal for differentiation along the gd T cell lineage. Lymph node development is also impaired in the absence of Ikaros. Ikaros null mice lack inguinal, axial, cervical and mesenteric lymph nodes, Peyer’s patches and lymphoid follicles in the gastrointestinal tract. Transplantation of wild type bone marrow into these mice repopulates the lymphoid compartment but fails to generate lymph node structures suggesting Ikaros is a critical factor in the temporally regulated formation of peripheral lymphatic centers during development ŽA. Nichogiannopoulou and K. Georgopoulos, unpublished results..
More severe effects on lymphoid differentiation are caused by an Ikaros dominant negative mutation Young Ikaros DN q ry mice appear to develop a normal lymphoid compartment. However, thymocytes and splenic T cells display an augmented proliferative response upon TCR stimulation. In addition, between 2 and 3 months of age thymocytes are highly enriched for CD4 q CD8 int TCR q and CD4 int CD8qTCRq cells, the transitional stages of differentiation during which thymic selection occurs. Moreover, clonal expansions of thymocytes with forbidden TCR specificities20 are frequently observed in these mutants. Together these findings suggest a deregulation in the outcome of negative selection during thymocyte differentiation. Three to sixmonth-old Ikaros DN q ry mice develop T cell
The role of Ikaros in hemopoietic precursors The presence of Ikaros mRNA at the earliest sites of hemopoiesis during ontogeny Žsee Ref 11 and T. Ikeda, in preparation. and in populations enriched for the earliest identifiable hemopoietic precursors3,4 prompted a more detailed analysis of the hemopoietic 122
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genitors ŽLiny c-kitq Sca1q Sca2q . express Helios. Pro-B cells have a low level of Helios expression which is further down-regulated in pre-B and mature B cells. Nevertheless, Helios expression persists in thymic and splenic T cell subpopulations. However, unlike Ikaros, Helios is not expressed in erythroid and myeloid lineages. Hemopoietic expression of Dedalos, the fourth family member, is restricted to bone marrow populations enriched for stem cells and early T cell precursors. These expression data hint at the possible involvement of Helios and Dedalos gene products in the early precursor defects observed in Ikaros DN mutants.
precursor compartment in both Ikaros mutants. Ikaros null bone marrow cellularity is reduced two fold, while splenic cellularity approximates normal levels in spite of the lack of B and T cells. Bone marrow is selectively depleted for immature ŽBFU-E . and mature ŽCFU-E . erythroid precursors but contains near normal numbers of granulocytic, monocytic ŽCFC-GrM . and multipotential ŽCFCmulti. precursors. Loss of Ikaros also reduces the number of spleen colony forming cells ŽCFU-S 14 ..27 Nevertheless Ikaros null bone marrow can radioprotect lethally irradiated recipients.28 Null bone marrow contains cells bearing the hemopoietic stem cell phenotype ŽLiny c-kit q Sca1q . 5,6 at normal frequencies and is able to provide long-term repopulation of erythroid and myeloid lineages in an irradiated host. Yet when co-injected with wild type bone marrow in a competitive repopulation assay,29,30 Ikaros null bone marrow cells fail to contribute to the hemopoietic compartment of the recipient. This latter finding suggests Ikaros null stem cells are either reduced in number or unable to compete effectively with wild type cells in the repopulation of recipients. These data strongly suggest an important yet not essential role for Ikaros in the engraftment, production or maintenance of hemopoietic stem cells or their immediate progeny ŽFigure 2.. The more severe defects manifested in the hemopoietic stem cell compartment of Ikaros DN homozygotes also compel a role for other factors which work in concert with Ikaros and against which DN Ikaros isoforms exert their effects in these processes ŽA. Nichogiannopoulou, in preparation..
Future directions Mice mutant for each of the Ikaros family members are currently being generated and studied to delineate the role of each gene in hemopoietic development. Distinct phenotypes resulting from each mutation will reveal essential and non-overlapping functions for Ikaros, Aiolos, Dedalos and Helios proteins. The presence or absence of novel phenotypes following genetic crossing of single mutants should reveal the degree of functional redundancy between these factors in hemopoietic lineage specification and homeostasis. The molecular mechanisms by which Ikaros exerts its function may relate in part to the transcriptional regulation of genes which are essential for lineage commitment and subsequent differentiation. Differential mRNA analysis between wild type and mutant Ikaros cell populations may identify genetic targets which when inappropriately expressed lead to the profound lymphoid phenotypes observed in Ikaros mutant mice. In mature lymphocytes, Ikaros provides an activation threshold which is overcome by TCR ligation. Changes in Ikaros-containing nuclear structures upon TCR engagement suggest a possible change in Ikaros function from regulating transcription to regulating DNA metabolism during cell cycle. The mechanisms by which Ikaros protein is targeted by TCR signaling cascades and through which Ikaros regulates stable propagation of the genome are currently being investigated. The mesodermal expression pattern of Ikaros is also being exploited to study the earliest events in embryonic and fetal hemopoiesis. By targeting the Lac Z reporter gene into the endogenous Ikaros locus we have a novel strategy for isolating the earliest hemopoietic populations and assessing their de-
Ikaros dimerization partners in the hemolymphoid system. Aiolos, Helios and Dedalos were identified as three novel Ikaros homologues which homo- and heterodimerize with Ikaros and each other ŽSee 3,31 and B.A. Morgan, unpublished results.. Aiolos and Helios proteins show extensive homology to Ikaros DNA-binding, dimerization and activation domains. Aiolos expression is restricted to the lymphoid lineages.3 During embryogenesis, Helios expression pattern resembles that of Ikaros. Helios is detected in day 8.5 yolk sac blood islands and day 9.5 fetal liver although at lower levels than those observed for Ikaros. Its expression in the fetal thymus is not detected until day 15, which is later than Ikaros detection. In the adult bone marrow populations enriched for stem cells ŽLiny c-kit q Sca1q . and lymphoid pro123
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10. Wang J, Nichogiannopoulou A, Wu L, Sun L, Sharpe A, Bigby M, Georgopoulos K Ž1996. Selective defects in the development of the fetal and adult lymphoid system in mice with an Ikaros null mutation. Immunity 5:537]549 11. Georgopoulos K, Bigby M, Wang J-H, Molnar A, Wu P, Winandy S, Sharpe A Ž1994. The Ikaros gene is required for the development of all lymphoid lineages. Cell 79:143]156 12. Kondo M, Weissman IL, Akashi K Ž1997. Identification of clonogenic common progenitors in mouse bone marrow. Cell 91:661]672 13. Wu L, Nichogiannopoulou A, Shortman K, Georgopoulos K Ž1997. Cell-autonomous defects in dendritic cell populations of Ikaros mutant mice point to a developmental relationship with the lymphoid lineage. Immunity 7:483]492 14. Ardavin C, Wu L, Li C, Shortman K Ž1993. Thymic dendritic cells and T cells develop simultaneously in the thymus from a common precursor population. Nature 362:761]763 15. Shortman K, Wu L Ž1996. Early T lymphocyte progenitors. Annu Rev Immunol 14:29]47 16. Wu L, Vremec D, Ardavin K, Winkel G, Suess IL, Maraskovsky E, Cook W, Shortman K Ž1995. Mouse thymus dendritic cells: kinetics of development and changes in surface markers during maturation. Eur J Immunol 25:418]425 17. Taccioli GE, Rathbun G, Shinkai Y, Oltz EM, Cheng H, Whitmore G, Stamato T, Jeggo P, Alt FW Ž1992. Activities involved in VŽD.J recombination. Curr Top Microbiol Immunol 182:107]114 18. Grosschedl R Ž1995. Higher-order nucleoprotein complexes in transcription: analogies with site-specific recombination ŽReview.. Curr Opin Cell Biol 7:362]370 19. Sleckman BP, Bardon CG, Ferrini R, Davidson L, Alt FW Ž1997. Function of the TCR alpha enhancer in alpha beta and gamma delta T cells. Immunity 7:505]515 20. Scherer MT, Ignatowicz L, Winslow GM, Kappler JW, Marrack P Ž1993. Superantigens: bacterial and viral proteins that manipulate the immune system. Annu Rev Cell Biol 9:101]128 21. Winandy S, Wu P, Georgopoulos K Ž1995. A dominant mutation in the Ikaros gene leads to rapid development of leukemia and lymphoma. Cell 83:289]299 22. Cavenee WK, Dryja TP, Phillips RA, Benedict WF, Godbout R, Gallie BL, Murphree AL, Strong LC, White RL Ž1983. Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature 305:779]784 23. Meltzer S, Yin J, Huang Y, McDaniel T, Newkirk C, Iseri O, Vogelstein B, Resau J Ž1991. Reduction to homozygosity involving p53 in esophageal cancers demonstrated by the polymerase chain reaction. Proc Natl Acad Sci USA 88:4976]4980 24. Ichii S, Horii A, Nakatsuru S, Furuyama J, Utsunomiya J, Nakamura Y Ž1992. Inactivation of both APC alleles in an early stage of colon adenomas in a patient with familial adenomatous polyposis ŽFAP.. Hum Mol Genet 1:387]390 25. Avitahl N, Friedrich C, Winandy S, Jones B, Ge Y, Georgopoulos K Ž1998. A macromolecular complex containing Ikaros controls T cell activation and chromosome homeostasis. Žsubmitted for publication. 26. Brown KE, Guest SS, Smale ST, Hahm K, Merkenschlager M, Fisher AG Ž1997. Association of transcriptionally silent genes with Ikaros complexes at centromeric heterochromatin. Cell 91:845]854 27. Till JE, McCulloch EA Ž1961. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res 14:213]222 28. Mulder AH, Visser JW Ž1987. Separation and functional analysis of bone marrow cells separated by rhodamine-123 fluorescence. Exp Hematol 15:99]104
velopmental potential and molecular composition. When combined with tissue explant experiments, this approach will ultimately seek to identify factors that induce Ikaros expression, an event which in the early embryo may be concomitant with mesoderm commitment to hemopoietic cell fate.
Acknowledgements We wish to thank T. Ikeda, J. Koipally, C. Kelley, N. Avitahl, S. Winandy and B. Morgan for constant valuable discussion and the communication of unpublished results. K. Georgopoulos is a Scholar of the Leukemia Society of America. M. Trevisan is an NCIC Terry Fox Junior Research Fellow supported with funds provided by the Terry Fox Run. The transgenic and other research were supported by an ACS grant to K. Georgopoulos and by a core grant from the Cutaneous Biology Research Center ŽShiseido Co. Ltd..
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