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Using conditional gene targeting in mice, we show that the chromatin remodeler Mi-2beta is crucial for different aspects of skin development. Early (E10.5) depletion of Mi-2beta in the developing ventral epidermis results in the delayed reduction of its suprabasal layers in late embryogenesis and to the ultimate depletion of its basal layer. Later (E13.5) loss of Mi-2beta in the dorsal epidermis does not interfere with suprabasal layer differentiation or maintenance of the basal layer, but induction of hair follicles is blocked. After initiation of the follicle, some subsequent morphogenesis of the hair peg may proceed in the absence of Mi-2beta, but production of the progenitors that give rise to the inner layers of the hair follicle and hair shaft is impaired. These results suggest that the extended self-renewal capacity of epidermal precursors arises early during embryogenesis by a process that is critically dependent on Mi-2beta. Once this process is complete, Mi-2beta is apparently dispensable for the maintenance of established repopulating epidermal stem cells and for the differentiation of their progeny into interfollicular epidermis for the remainder of gestation. Mi-2beta is however essential for the reprogramming of basal cells to the follicular and, subsequently, hair matrix fates.

Hematopoiesis is the developmental process by which all blood and immune cells are generated. A decade-old scheme has supported an early and complete separation of the erythro-myeloid from the lymphoid lineages. Recent advances have re-drawn this map, separating lymphoid and myeloid from erythroid programs early in development. Subsequently, the fate restriction of both the lympho-myeloid and the erythro-megakaryocyte progenitors is dependent on Ikaros and its associated chromatin regulators. Genetic studies of this family of nuclear factors are now providing unique insight into the functional molecular signatures that bestow plasticity to the hematopoietic stem cell and its early progeny.

Ikaros DNA-binding proteins are critical for the development of lymphocytes and other hematopoietic lineages, but it remains unclear how they cooperate with other regulators of signaling and transcription to achieve ordered gene expression during development. Here, we show that Ikaros proteins regulate the pre-BCR component lambda5 in a stage-specific manner. In pre-BI cells, Ikaros modulated lambda5 expression in competition with the transcriptional activator EBF. This required Ikaros binding to the Igll1 (lambda5) promoter and was abolished either by mutation of the Ikaros DNA-binding domain or by deletion of a single Ikaros site from the Igll1 promoter. At the transition from the pre-BI to pre-BII stage, the expression of the Ikaros family member Aiolos was upregulated and required for the efficient silencing of Igll1. Aiolos expression was controlled by pre-BCR signals via the adaptor protein SLP-65. Thus, pre-BCR signaling regulates Aiolos and the silencing of Igll1 via a developmental-stage-specific feedback loop.

Ikaros is expressed in early hematopoietic progenitors and is required for lymphoid differentiation. In the absence of Ikaros, there is a lack of markers defining fate restriction along lympho-myeloid pathways, but it is unclear whether formation of specific progenitors or expression of their markers is affected. Here we use a reporter based on Ikaros regulatory elements to separate early progenitors in wild-type and Ikaros-null mice. We found previously undetected Ikaros-null lympho-myeloid progenitors lacking the receptor tyrosine kinase Flt3 that were capable of myeloid but not lymphoid differentiation. In contrast, lack of Ikaros in the common myeloid progenitor resulted in increased formation of erythro-megakaryocytes at the expense of myeloid progenitors. Using this approach, we identify previously unknown pivotal functions for Ikaros in distinct fate 'decisions' in the early hematopoietic hierarchy.

ICF (Immunodeficiency, Centromeric instability and Facial anomalies) syndrome is a rare autosomal recessive disease caused by mutations in the DNA methyltransferase gene DNMT3B. To investigate the function of Dnmt3b in mouse development and to create animal models for ICF syndrome, we have generated three mutant alleles of Dnmt3b in mice: one carrying a deletion of the catalytic domain (null allele) and two carrying ICF-like missense mutations in the catalytic domain. The Dnmt3b null allele results in embryonic lethality from E14.5 to E16.5 with multiple tissue defects, including liver hypotrophy, ventricular septal defect and haemorrhage. By contrast, mice homozygous for the ICF mutations develop to term and some survive to adulthood. These mice show phenotypes that are reminiscent of ICF patients, including hypomethylation of repetitive sequences, low body weight, distinct cranial facial anomalies and T cell death by apoptosis. These results indicate that Dnmt3b plays an essential role at different stages of mouse development, and that ICF missense mutations cause partial loss of function. These mutant mice will be useful for further elucidation of the pathogenic and molecular mechanisms underlying ICF syndrome.

Integrin-linked kinase (ILK) is a serine/threonine kinase that is important in cell-matrix interactions and cell signaling. To examine the role of ILK in leukocyte trafficking and survival, we generated T cell-specific ILK knockouts by breeding ILK(flox/flox) mice to transgenic mice expressing Cre recombinase under control of the Lck proximal promoter. Thymic T cells from Lck-Cre(+)/ILK(flox/flox) mice had a marked reduction (>95%) in ILK protein levels. Thymic cellularity was comparable in 3- to 4-week-old mice, but a threefold diminution of thymic T cells became evident by 6 to 8 weeks of age in the T cell-specific ILK knockout mice due to increased cell death of double-positive (DP) T cells. Analysis of peripheral T cells by quantitative PCR and by breeding Lck-Cre(+)/ILK(flox/flox) mice to a YFP-transgenic reporter strain demonstrated an approximate 20-fold enrichment of ILK-competent cells, suggesting these cells have a competitive advantage in trafficking to and/or survival in peripheral lymphatic organs. We explored mechanisms related to altered cell trafficking and survival that might explain the decreases in thymic cellularity and enrichment for ILK-competent cells in the spleen and lymph nodes. We observed a >50% reduction in chemotaxis of ILK-deficient T cells to the chemokines CXCL12 (stromal cell-derived factor [SDF]-1alpha) and CCL19 (macrophage inflammatory protein [MIP]-3beta), as well as enhanced apoptosis of ILK-deficient cells upon stress. Signaling studies in ILK-deficient T cells demonstrated diminished phosphorylation of Akt on the activating phosphorylation site, Ser 473, and a concordant decrease in Akt kinase activity following stimulation with the chemokine SDF-1. Rac1 activation was also markedly diminished in ILK-deficient T cells following chemokine stimulation. These data extend the role of ILK to immune-cell trafficking and survival via modulation of Akt- and Rac-dependent substrates, and have implications for cell recruitment in both homeostatic and pathological processes.

Ikaros plays a key role in lymphocyte development and homeostasis by both potentiating and repressing gene expression. Here we show that Ikaros interacts with components of the SUMO pathway and is SUMOylated in vivo. Two SUMOylation sites are identified on Ikaros whose simultaneous modification results in a loss of Ikaros' repression function. Ikaros SUMOylation disrupts its participation in both histone deacetylase (HDAC)-dependent and HDAC-independent repression but does not influence its nuclear localization into pericentromeric heterochromatin. These studies reveal a new dynamic way by which Ikaros-mediated gene repression is controlled by SUMOylation.

Changes in chromatin structure underlie the activation or silencing of genes during development. The chromatin remodeler Mi-2beta is highly expressed in thymocytes and is presumed to be a transcriptional repressor because of its presence in the nucleosome remodeling deacetylase (NuRD) complex. Using conditional inactivation, we show that Mi-2beta is required at several steps during T cell development: for differentiation of beta selected immature thymocytes, for developmental expression of CD4, and for cell divisions in mature T cells. We further show that Mi-2beta plays a direct role in promoting CD4 gene expression. Mi-2beta associates with the CD4 enhancer as well as the E box binding protein HEB and the histone acetyltransferase (HAT) p300, enabling their recruitment to the CD4 enhancer and causing histone H3-hyperacetylation to this regulatory region. These findings provide important insights into the regulation of CD4 expression during T cell development and define a role for Mi-2beta in gene activation.

Ikaros is a key regulator of lymphocyte proliferative responses. Inactivating mutations in Ikaros cause antigen-mediated lymphocyte hyperproliferation and the rapid development of leukemia and lymphoma. Here we show that Ikaros's ability to negatively regulate the G(1)-S transition can be modulated by phosphorylation of a serine/threonine-rich conserved region (p1) in exon 8. Ikaros phosphorylation in p1 is induced during the G(1)-S transition. Mutations that prevent phosphorylation in p1 increase Ikaros's ability to impede cell cycle progression and its affinity for DNA. Casein kinase II, whose increased activity in lymphocytes leads to transformation, is a key player in Ikaros p1 phosphorylation. We thus propose that Ikaros's activity as a regulator of the G(1)-S transition is controlled by phosphorylation in response to signaling events that down-modulate its DNA binding activity.

Antigenic encounter generates long-term immunity sustained by long-lived high affinity plasma cells resident in the bone marrow (BM). Here we show that the Ikaros family member, Aiolos, is specifically required for the generation of these plasma cells. Failure to generate high affinity plasma cells in the BM and to sustain serum antibody titers is apparent after both primary and secondary immunization of Aiolos(-)(/)(-) mice with a range of hapten concentrations. Chimera reconstitutions demonstrate that the BM plasma cell defect is B cell intrinsic. Lack of Aiolos does not alter expression of any of the previously described factors required for general plasma cell differentiation. No defect in somatic hypermutation, the generation of memory B cells, or short-lived high affinity plasma cells in the spleen was observed upon rechallenge. These studies support a model by which the high affinity plasma cell population in the BM undergoes a unique differentiation program that is dependent on Aiolos.

Regulated expression of Ikaros is critical for normal hemopoiesis and lymphocyte development. To elucidate the mechanisms underlying transcription of Ikaros, tissue-specific DNase I-hypersensitive sites (DHS) were mapped throughout the Ikaros locus, and several promoters were identified. The activity of these regulatory regions was elucidated using an enhanced green fluorescent protein (EGFP) reporter in transgenic mice. Two genomic fragments, each containing a distinct promoter and its associated DHS cluster, were found to be active in the myeloid (DHS-C2 and DHS-C3) and B-cell (DHS-C3) lineages. Although neither of these regulatory regions was active within the majority of differentiating thymocytes and mature T cells, the DHS-C3 region was active at the earliest stages (DN1-DN3) of T-cell differentiation. However, when the DHS-C3 region was combined with the downstream intronic DHS-C6 cluster, its activity was maintained and raised to higher levels at subsequent stages of T-cell differentiation. This combination of regulatory elements provided reporter expression that closely resembles that of endogenous Ikaros during hemo-lymphopoiesis, and it decreased (but did not alleviate) position effect variegation within the expressing cell types.

Here we show that mice lacking the zinc finger transcription factor Aiolos develop the symptoms of human systemic lupus erythematosus (SLE), which is characterized by the production of anti-dsDNA Ab and immune complex-mediated glomerulonephritis. This finding indicates that normal Aiolos function is necessary to maintain immune homeostasis and suppress the development of systemic autoimmune disease and implicates Aiolos as a possible candidate gene for SLE. Interestingly, Aiolos-null mice can no longer mount autoimmune reactions and completely fail to develop SLE when they are deficient for the B cell-specific transcription coactivator OBF-1. The lack of OBF-1 reverses several Aiolos mutant mouse phenotypes, such as B cell hyperproliferation, high expression of activation marker on B cells, and spontaneous germinal center formation. Unexpectedly, B cell development at the immature B cell stage is severely impaired in the bone marrow of Aiolos/OBF-1 double-deficient mice, demonstrating the key role of these factors in the transition from pre-B to immature B cells. Our results indicate that B cells play a crucial role in the development of SLE in Aiolos mutant mice and might be useful for the strategy of SLE treatment.

Ikaros family members are important regulatory factors in lymphocyte development. Here we show that Ikaros may play an important role in CD4 versus CD8 lineage commitment decisions by demonstrating: (1) that it binds to regulatory elements in the endogenous CD8alpha locus in vivo using thymocyte chromatin immunoprecipitations, (2) that Ikaros suppresses position effect variegation of transgenes driven by CD8 regulatory elements, and (3) that mice with reduced levels of Ikaros and Aiolos show an apparent increase in CD4 populations with immature phenotype, i.e., cells that failed to activate the CD8alpha gene locus. We propose that Ikaros family members function as activators of the CD8alpha gene locus and that their associated activities are critical for appropriate chromatin remodeling transitions during thymocyte differentiation and lineage commitment.

Ikaros is a key regulator of the hemo-lymphoid system in which it is presumed to function by both potentiating and repressing gene expression. Repression is mediated through two independent domains at the N and C terminus of the protein, both of which can independently recruit the corepressors Mi-2beta, Sin3A, and Sin3B and the Class I histone deacetylases 1 and 2; the N-terminal domain can also associate with the corepressor CtBP. Here we describe a detailed dissection of these two domains and identify the minimal repression modules and the corepressor requirements for their activity. Based on these studies, we describe mutations in a full-length Ikaros protein that abrogate interactions with each of the identified corepressors and abolish the protein's function as a repressor. Finally, we show that, barring CtBP, the Ikaros family members Aiolos, Helios, and Eos can associate with all of the identified corepressors of Ikaros including its newly identified interactors, Class II HDACs.

Ikaros and Aiolos are Kruppel zinc finger proteins that play key roles in hemo-lymphoid development and homeostasis. We have previously shown that they can repress transcription through the recruitment of histone deacetylases (HDACs). Here, we provide the first functional evidence that these proteins can also repress gene function in a manner that does not require deacetylase activity. This functionality can be attributed in part to Ikaros interactions with the HDAC-independent corepressor, C-terminal binding protein (CtBP). However, mutations that block Ikaros-CtBP interactions do not abolish Ikaros's repression activity, implicating the involvement of additional corepressors. Consistent with this expectation, we show that Ikaros can interact with a CtBP-interacting protein (CtIP), which has also been linked to a deacetylase-independent strategy of repression. Despite being a CtBP interactor, CtIP's association with Ikaros does not require CtBP but instead relies upon its Rb interaction domain. Significantly, Ikaros can interact with Rb, which itself can repress gene function in a deacetylase-independent manner. A mutation in Ikaros that abrogates CtIP interactions significantly reduces repression, and a double mutation that prevents interaction with both CtIP and CtBP even further alleviates repression. Finally, we show that CtIP and CtBP can interact with the general transcription factors, TATA binding protein and transcription factor IIB, which suggests a possible mechanism for their deacetylase-independent mode of repression.

Ikaros is essential for the normal development and regulated proliferation of lymphoid cells. In lymphocytes, Ikaros exists as an integral component of chromatin-remodeling complexes, including the Mi-2beta/nucleosome remodeling and deacetylation complex (NuRD) complex. It is expected that Ikaros, together with these associated activities effects repression, but here we show that they may also potentiate gene expression in cycling cells. Ikaros cannot activate transcription by itself; instead, it enhances the activity of both weak and strong activators. For this role in potentiation, Ikaros requires its DNA binding and dimerization domains. The DNA binding and dimerization properties of Ikaros are also responsible for its targeting to pericentromeric heterochromatin (PC-HC). Significantly, Ikaros mutants with altered specificity for DNA binding that are unable to localize to PC-HC are incapable of stimulating transcription from reporters bearing their cognate sites. Thus, potentiation of gene expression by Ikaros correlates strongly with its ability to localize to PC-HC in combination with the chromatin remodeler Mi-2beta.

The regulated production of several terminally differentiated cell types of the blood and immune systems (haematopoiesis) has been the focus of many studies on cell-fate determination. Chromatin and the control of its structure have been implicated in the regulation of cell-fate decisions and in the maintenance of the determined states. Here, I review advances in the field, emphasizing the potential role of chromatin in lineage commitment and differentiation. In this context, I discuss Ikaros, an essential regulator of lymphocyte development and an integral component of a functionally diverse chromatin remodelling network that operates from the early stages of haematopoiesis to the mature lymphocytes.

Mink cell focus-inducing (MCF) viruses induce T-cell lymphomas in AKR/J strain mice. MCF 247, the prototype of this group of nonacute murine leukemia viruses, transforms thymocytes, in part, by insertional mutagenesis and enhancer-mediated dysregulation of cellular proto-oncogenes. The unique 3' (U3) regions in the long terminal repeats of other murine leukemia viruses contain transcription factor binding sites known to be important for enhancer function and for the induction of T-cell lymphomas. Although transcription factor binding sites important for the biological properties of MCF 247 have not been identified, pathogenesis studies from our laboratory suggested to us that binding sites for Ikaros, a lymphoid-cell-restricted transcriptional regulator, affect the biological properties of MCF 247. In this report, we demonstrate that Ikaros binds to predicted sites in U3 sequences of MCF 247 and that site-directed mutations in these sites greatly diminish this binding in vitro. Consistent with these findings, ectopic expression of Ikaros in murine cells that do not normally express this protein significantly increases transcription from the viral promoter in transient gene expression assays. Moreover, site-directed mutations in specific Ikaros-binding sites reduce this activity in T-cell lines that express Ikaros endogenously. To determine whether the Ikaros-binding sites are functional in vivo, we inoculated newborn mice with a variant MCF virus containing a mutant Ikaros-binding site. The variant virus replicated in thymocytes less efficiently and induced lymphomas with a delayed onset compared to the wild-type virus. These data are consistent with the hypothesis that the Ikaros-binding sites in the U3 region of MCF 247 are functional and cooperate with other DNA elements for optimal enhancer function in vivo.

The lck gene encodes a protein-tyrosine kinase that plays a key role in signaling mediated through T cell receptor (TCR) and pre-TCR complexes. Transcription of the lck gene is regulated by two independent promoter elements: the proximal and distal promoters. Previous studies employing transgenic mice demonstrated that the sequence between -584 and -240 from the transcription start site in the mouse lck proximal promoter is required for its tissue-specific expression in the thymus. In this study, we demonstrate that a Krüppel-like zinc finger protein, mtbeta (BFCOL1, BERF-1, ZBP-89, ZNF148), previously cloned as a protein that binds to the CD3delta gene enhancer, binds to the -365 to -328 region of the lck proximal promoter. mtbeta is ubiquitously expressed in various cell lines and mouse tissues. Overexpressed mtbeta is more active in T-lineage cells than B-lineage cells for transactivating an artificial promoter consisting of the mtbeta binding site and a TATA box. Activity of the lck proximal promoter was significantly impaired by mutating the mtbeta binding site or by reducing mtbeta protein expression level by using antisense mRNA. Our results indicate that mtbeta activity is regulated in a tissue-specific manner and that mtbeta is a critical transactivator for the lck proximal promoter.

Most splenic B cells in mice that lack Aiolos are mature IgD(hi)IgM(lo) follicular lymphocytes, suggesting that maturation signals delivered via the BCR are enhanced in the absence of Aiolos. The enhanced maturation of follicular B cells is accompanied by the absence of MZ B lymphocytes and the downregulation of CD21 expression in follicular B cells, all of which depend on the generation of signals via Btk, which is in epistasis to Aiolos. The inverse relationship between the strength of BCR signaling and MZ B cell development is supported by an examination of MZ B cells in CD21 null mice. These data support the view that antigens (in contrast to "tonic" signals) drive the development of naive B cells.

The antigen presenting dendritic cells (DC) found in mouse and human lymphoid tissues are heterogeneous. Several subsets of mature DC have been described and these may correspond to distinct lineages. In this review, we present evidence obtained from a series of studies on the lineage origin of DC. This evidence points to the existence of at least three pathways for DC development, namely one from myeloid progenitors, a second from lymphoid progenitors and the third for Langerhans cells from precursors whose relationship to myeloid or lymphoid cell types is not yet clearly defined.

We have previously shown that Ikaros can repress transcription through the recruitment of histone deacetylase complexes. Here we provide evidence that Ikaros can also repress transcription through its interactions with the co-repressor, C-terminal binding protein (CtBP). CtBP interacts with Ikaros isoforms through a PEDLS motif present at the N terminus of these proteins but not with homologues like Aiolos which lack this motif. Mutations in Ikaros that prevent CtBP interactions reduce its ability to repress transcription. CtBP interacts with Sin3A but not with the Mi-2 co-repressor and it represses transcription in a manner that is independent of histone deacetylase activity. These data strongly suggest that CtBP contributes to a histone deacetylase activity independent mechanism of repression by Ikaros. Finally, we show that the viral oncoprotein E1A, which binds to CtBP, also shows a strong association with Ikaros. This Ikaros-E1A interaction may underlie Ikaros's decreased ability to repress transcription in E1A transformed cells.

The molecular and cellular requirements for the development of different populations of human dendritic cells (DC) were studied. Conditions were defined that support DC production from lymphoid progenitors but that fail to induce DC formation from peripheral monocytes. The production of these lymphoid-related DC was severely blocked when hematopoietic progenitors overexpressed Ik7, a mutant dominant-negative Ikaros protein. In contrast, Ik7 did not block the formation of DC in conditions supporting the development of monocyte-derived DC. Furthermore, Ik7 did not block the formation of monocyte/macrophages and enhanced granulopoiesis. One of the molecular mechanisms mediated by Ik7 appears to be down-regulation of the flt3-receptor mRNA. Thus, distinct signals control the formation of DC demonstrating that some aspects of DC diversity are determined in part by distinct molecular cues at the hematopoietic level. (Blood. 2000;95:128-137)

Here we provide evidence that the Ikaros family of DNA binding factors is critical for the activity of hemopoietic stem cells (HSCs) in the mouse. Mice homozygous for an Ikaros null mutation display a >30-fold reduction in long-term repopulation units, whereas mice homozygous for an Ikaros dominant negative mutation have no measurable activity. The defect in HSC activity is also illustrated by the ability of wild-type marrow to repopulate unconditioned Ikaros mutants. A progressive reduction in multipotent CFU-S(14) (colony-forming unit-spleen) progenitors and the earliest erythroid-restricted precursors (BFU-E [burst-forming unit-erythroid]) is also detected in the Ikaros mutant strains consistent with the reduction in HSCs. Nonetheless, the more mature clonogenic erythroid and myeloid precursors are less affected, indicating either the action of a compensatory mechanism to provide more progeny or a negative role of Ikaros at later stages of erythromyeloid differentiation. In Ikaros mutant mice, a decrease in expression of the tyrosine kinase receptors flk-2 and c-kit is observed in the lineage-depleted c-kit(+)Sca-1(+) population that is normally enriched for HSCs and may in part contribute to the early hemopoietic phenotypes manifested in the absence of Ikaros.

T cell differentiation relies on pre-T cell receptor (TCR) and TCR signaling events that take place at successive steps of the pathway. Here, we show that two of these T cell differentiation checkpoints are regulated by Ikaros. In the absence of Ikaros, double negative thymocytes can differentiate to the double positive stage without expression of a pre-TCR complex. Subsequent events in T cell development mediated by TCR involving transition from the double positive to the single positive stage are also regulated by Ikaros. Nonetheless, in Ikaros-deficient thymocytes, the requirement of pre-TCR expression for expansion of immature thymocytes as they progress to the double positive stage is still maintained, and the T cell malignancies that invariably arise in the thymus of Ikaros-deficient mice are dependent on either pre-TCR or TCR signaling. We conclude that Ikaros regulates T cell differentiation, selection, and homeostasis by providing signaling thresholds for pre-TCR and TCR.

Here we show that the lymphoid lineage-determining factors Ikaros and Aiolos can function as strong transcriptional repressors. This function is mediated through two repression domains and is dependent upon the promoter context and cell type. Repression by Ikaros proteins correlates with hypo-acetylation of core histones at promoter sites and is relieved by histone deacetylase inhibitors. Consistent with these findings, Ikaros and its repression domains can interact in vivo and in vitro with the mSin3 family of co-repressors which bind to histone deacetylases. Based on these and our recent findings of associations between Ikaros and Mi-2-HDAC, we propose that Ikaros family members modulate gene expression during lymphocyte development by recruiting distinct histone deacetylase complexes to specific promoters.

The Ikaros gene family encodes zinc finger DNA-binding proteins essential for lineage determination and control of proliferation in the lymphoid system. Here, we report that, in the nucleus of a T cell, a major fraction of Ikaros and Aiolos proteins associate with the DNA-dependent ATPase Mi-2 and histone deacetylases, in a 2 MD complex. This Ikaros-NURD complex is active in chromatin remodeling and histone deacetylation. Upon T cell activation, Ikaros recruits Mi-2/HDAC to regions of heterochromatin. These studies reveal that Ikaros proteins are capable of targeting chromatin remodeling and deacetylation complexes in vivo. We propose that the restructuring of chromatin is a key aspect of Ikaros function in lymphocyte differentiation.

T cell activation involves the sustained accumulation of T cell receptor (TCR) and IL-2 receptor (IL-2R) mediated signaling events that promote cell cycle entry and progression. The Ikaros family of nuclear factors regulate this process by providing thresholds overcome by receptor signaling. T cells with reduced levels of Ikaros activity require fewer TCR engagement events for activation, exhibit a greater proliferative response to IL-2, and are less sensitive to inhibitors of TCR and IL-2R signaling. Upon T cell activation, Ikaros proteins localize in a higher-order chromatin structure where they colocalize with components of the DNA replication machinery. Proliferating T cells with reduced Ikaros activity display chromosome abnormalities. We propose that participation of Ikaros in higher-order chromatin structures controls cell cycle transitions and restricts DNA replication.

Aiolos encodes a zinc finger DNA-binding protein that is highly expressed in mature B cells and is homologous to Ikaros. In the periphery of mice homozygous for an Aiolos-null mutation, B cells exhibit an activated cell surface phenotype and undergo augmented antigen receptor (BCR)-mediated in vitro proliferative responses, even at limiting amounts of stimulant. In vivo, T cell-dependent B cell responses, including the formation of germinal centers and elevated serum IgG and IgE, are detected in Aiolos-deficient mice in the absence of immunization. Auto-antibodies and development of B cell lymphomas are frequently seen among aging Aiolos mutants. In sharp contrast to conventional B cells, B cells of the peritoneum, of the marginal zone, and the recirculating bone marrow population are greatly reduced.

Thymic dendritic cells (DC) mediate negative selection at a relatively late stage of the T-cell developmental pathway. We present evidence that the development of thymic DC and of T-lineage cells is linked via a common precursor at an early stage of thymocyte development. T-lineage precursor populations from the adult mouse thymus, prior to T-cell receptor gene rearrangement, display a capacity to produce DC as well as T cells in the thymus, and are very efficient precursors of DC in culture. These lymphoid/DC precursors have little capacity to form myeloid cells, indicating that thymic DC are a lymphoid-related rather than myeloid-related lineage. In contrast to myeloid-related DC, granulocyte-macrophage colony-stimulating factor is not required for the development of these lymphoid-related DC in vivo or in vitro. DC can develop in mutant mice lacking mature T cells, provided the common precursors are present. However, in mutant mice lacking functional Ikaros transcription factors, there are deficiencies in lymphoid precursor cells, in mature lymphoid cells and in DC.

Background: Normal hematopoietic development depends on the activity of the Ikaros transcription factor, which contains distinct zinc-finger domains that mediate DNA binding and protein dimerization. Mice homozygous for a transgene encoding a dominant-negative version of Ikaros that lacks the DNA-binding domain but not the dimerization domain have a more severe phenotype than Ikaros null mice. This observation suggests the presence of factor(s) that can dimerize with Ikaros and partially complement its function. One previously identified factor, Aiolos, probably serves this role in the lymphoid system; a related factor involved in hematopoietic progenitors remains unknown, however., Results: Here, we describe the cloning of an Ikaros-related gene, Helios. Analysis of the primary sequences of Helios, Ikaros and Aiolos revealed that the DNA-binding, transcriptional activation and dimerization domains are functionally conserved. Helios activated transcription from Ikaros DNA-binding sites and could dimerize with itself, Ikaros or Aiolos. Expression of Helios was detected in the earliest hematopoietic sites of the embryo, in hematopoietic stem cells in the adult and was subsequently restricted to a subset of cells in the T cell lineage. Helios co-localized with Ikaros and Aiolos proteins in macromolecular nuclear structures and formed stable complexes in vivo with the dominant-negative version of Ikaros., Conclusions: Distinct but overlapping expression patterns of members of the Ikaros gene family during hematopoiesis might result in the formation of different multimeric complexes that have specific roles in lineage progression. The preferential expression of Helios in the earliest stages of hematopoiesis suggests that this gene functions predominantly in early progenitors.

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.

The transcription factor Ikaros is a major determinant of lymphocyte differentiation. Mice homozygous for an Ikaros dominant-negative (DN-/-) mutation lack all cells of lymphoid origin, including T, B, and natural killer (NK) cells. Mice homozygous for an Ikaros null allele lack B and NK cells but display specific defects in T lymphocytes. Nonetheless, both Ikaros mutant lines make an excess of monocytes and macrophages. Here we report that the production of subsets of antigen-presenting dendritic cells (DCs) is also defective. By constructing bone marrow chimeras, we demonstrate that the Ikaros-mediated defect in lymphocytes and DCs is intrinsic to their precursors and is not environment dependent. The selective defects in DCs manifested with the Ikaros null mutation suggest a tight linkage between development of T cells and CD8alpha+ DCs. The complete lack of DCs in the lymphoid organs of Ikaros DN-/- micke points to an essential role for the Ikaros gene family in the development of all DCs.

Development of the lymphoid system is dependent on the activity of zinc finger transcription factors encoded by the Ikaros gene. Differences between the phenotypes resulting from a dominant-negative and a null mutation in this gene suggest that Ikaros proteins act in concert with another factor with which they form heterodimers. Here we report the cloning of Aiolos, a gene which encodes an Ikaros homologue that heterodimerizes with Ikaros proteins. In contrast to Ikaros--which is expressed from the pluripotent stem cell to the mature lymphocyte--Aiolos is first detected in more committed progenitors with a lymphoid potential and is strongly up-regulated as these differentiate into pre-T and pre-B cell precursors. The expression patterns of Aiolos and Ikaros, the relative transcriptional activity of their homo- and heteromeric complexes, and the dominant interfering effect of mutant Ikaros isoforms on Aiolos activity all strongly suggest that Aiolos acts in concert with Ikaros during lymphocyte development. We therefore propose that increasing levels of Ikaros and Aiolos homo- and heteromeric complexes in differentiating lymphocytes are essential for normal progression to a mature and immunocompetent state.

Intimate interactions between multipotential hemopoietic stem cells and their microenvironment work towards redefining the identity and the differentiative fate of these primitive cells. Molecular cues delivered by the microenvironment frequently act in an instructive fashion by initiating intracellular signaling pathways that ultimately target a select group of transcription factors. These transcriptional regulators in turn trigger a cascade of genetic changes that ultimately determine the course of the cells during differentiation. Gene inactivation studies on the PU.1, Ikaros and GATA-3 genes have revealed that their encoded factors are essential for the earliest commitment step into the B and T lymphoid lineages.

The Ikaros gene, which encodes a family of hemopoietic-specific zinc finger proteins, is described as a central regulator of lymphocyte differentiation. During fetal development, it is required at the earliest stage of T cell and B cell specification. In the adult, however, lymphoid lineages rely on Ikaros at distinct phases of their development. Its activity is essential for the generation of B cell but not of T cell precursors, although the differentiation of the latter is not normal. A significant increase in CD4 thymocytes and their immediate precursors is detected, and because these cells lack markers that correlate with positive selection, a deregulation in their maturation process is suggested. Furthermore, Ikaros-null thymocytes hyperproliferate in response to T cell receptor (TCR) signaling; within days after their appearance in the thymus, clonally expanding populations are detected. Deregulated TCR-mediated responses and the fast kinetics of tumor development in these mutant thymocytes implicate Ikaros as a central tumor suppressor gene for the T cell lineage. In addition, lack of natural killer cells and selective defects in gamma delta T cells and dendritic antigen-presenting cells point to Ikaros as an essential factor for the establishment of early branchpoints of the T cell pathway. The dominant interference activity of Ikaros isoforms unable to bind DNA and their effects in lymphocyte development suggest that Ikaros works in concert with other factors. The role of Aiolos, a lymphoid-restricted and structurally related gene, in lymphoid differentiation is discussed. A model is proposed that defines Ikaros as the backbone of a complex regulatory protein network that controls cell fate decisions and regulates homeostasis in the hemo-lymphoid system. Changes in this regulatory network may reflect differentiation and proliferation adjustments made in hemo-lymphoid progenitors and precursors as they give rise to the cells of our immune system.

Mice homozygous for an Ikaros null mutation display distinct defects in the development of fetal and adult lymphocytes. Fetal T lymphocytes, and fetal and adult B lymphocytes and their earliest progenitors are absent. Postnatally, hematopoietic stem cells give rise to thymocyte precursors that undergo aberrant differentiation into the CD4 lineage and clonal expansion. The lack of NK cells and some gamma delta T cell subsets and a large reduction in thymic dendritic APCs suggest that Ikaros is essential for establishing early branch points in the postnatal T cell pathway. The lymphoid defects detected in Ikaros null mice reveal critical molecular differences between fetal and postnatal hematopoietic progenitors that dictate their ability to give rise to T cells. These studies also establish Ikaros as a tumor suppressor gene acting during thymocyte differentiation. Phenotypic comparison of this null mutation with a severe dominant-negative Ikaros mutation identifies molecular redundancy in the postnatal hemolymphoid system.

The Ikaros gene is essential for lymphoid lineage specification. As previously reported, mice homozygous for a mutation in the Ikaros DNA-binding domain fail to generate mature lymphocytes as well as their earliest described progenitors. In addition, our studies with mice heterozygous for this mutation establish the Ikaros gene as an essential regulator of T cell proliferation. Thymocytes display augmented TCR-mediated proliferative responses, and peripheral T cells are autoproliferative. A general lymphoproliferation precedes the T cell leukemia and lymphoma that rapidly develop in all heterozygotes. The first step toward leukemic transformation occurs within the maturing thymocyte population and is demarcated by clonal expansions and loss of the single Ikaros wild-type allele. From these studies, we propose that within developing and mature T lymphocytes, distinct thresholds of Ikaros activity are required to regulate proliferation. A decrease in Ikaros activity below the first threshold causes the rapid accumulation of T lymphoblasts, whereas a further decrease leads to neoplastic transformation.

The Ikaros gene encodes a family of early hematopoietic- and lymphocyte-restricted transcription factors. Mice homozygous for a germline mutation in the Ikaros DNA-binding domain lack not only T and B lymphocytes and natural killer cells but also their earliest defined progenitors. In contrast, the erythroid and myeloid lineages were intact in these mutant mice. We propose that Ikaros promotes differentiation of pluripotential hematopoietic stem cell(s) into the lymphocyte pathways. In the absence of a functional Ikaros gene, these stem cells are exclusively diverted into the erythroid and myeloid lineages.

Characterization of the transcription control regions for most genes encoding the TCR-CD3 complex has been followed by identification of DNA-binding factors, some of which are restricted to lymphoid or T-lineage cells. The sequential expression of these factors is linked with the appearance of stage-specific T-cell markers, thus controlling the commitment of the elusive lymphoid progenitor cell and its progression along the T-lineage. Here, Hans Clevers, Mariëtte Oosterwegel and Katia Georgopoulos discuss recent progress made in the recognition of transcription factors that regulate these early events in T-cell ontogeny.

In a screen for transcriptional regulators that control differentiation into the T cell lineage, a complementary DNA was isolated encoding a zinc finger protein (Ikaros) related to the Drosophila gap protein Hunchback. The Ikaros protein binds to and activates the enhancer of a gene encoding an early T cell differentiation antigen, CD3 delta. During development, Ikaros messenger RNA was first detected in the mouse fetal liver and the embryonic thymus when hematopoietic and lymphoid progenitors initially colonize these organs; no expression was observed in the spleen or the bone marrow. The pattern of Ikaros gene expression and its ability to stimulate CD3 delta transcription support the model that Ikaros functions in the specification and maturation of the T lymphocyte.

CD3 is a multi-subunit complex of proteins noncovalently associated with the T cell receptor (TcR) for antigen. Considerable evidence indicates a role for CD3 molecules in the transduction of activation signals in T cells. The murine CD3 delta gene encodes a 0.7-kb transcript present in mature T cells. Here we report the characterization of several additional CD3 delta transcripts; two nuclear transcripts, 4-4.5 kb in size, and two predominamtly cytoplasmic transcripts of 1.5 kb and 2.5 kb. Both T lymphoma cell lines and normal thymocytes express the 1.5-kb and 2.5-kb CD3 delta transcripts. These cytoplasmic transcripts have long 3'-untranslated sequences which extend beyond the polyadenylation site of the predominant 0.7-kb transcript. The protein synthesis inhibitor cycloheximide (CHX) increases the expression of all three cytoplasmic CD3 delta transcripts, indicating that their level of expression may be regulated by a labile inhibitor protein(s). The CHX elicited increase in CD3 delta mRNA appears to result from post-transcriptional events since the rate of CD3 delta gene transcription remains constant. In contrast to CHX, the calcium ionophore A23187 increases the rate of CD3 delta gene transcription and, like CHX, also increases the level of cellular CD3 delta mRNA. The immunosuppressive agent cyclosporin A inhibits A23187-mediated stimulation of transcription, but has no effect on the CHX-mediated induction of CD3 delta mRNA. We conclude that both transcriptional and post-transcriptional mechanisms can regulate the amount of all three cytoplasmic CD3 delta transcripts.

The existence of cross-reactivity between Klebsiella antigens and cells from donors who are HLA-B27 positive and exhibit ankylosing spondylitis (AS) has been reinvestigated. Cells and antisera from different laboratories have been tested together using simultaneously microcytoxicity, chromium release and enzyme linked immunosorbent assays (ELISA). No reproducible interaction has been found. Mitogenic stimulation did not induce cross-reactivity and 'transformation' of B27+AS- cells by Klebsiella culture supernatants failed. Two transformed cell lines from B27+ AS+ donors exhibited specific cross reaction with two anti-Klebsiella antisera but only by chromium release. Immunoprecipitation with these cells and antisera showed the absence of any AS+ -specific antigen. It is concluded that the involvement of Klebsiella in ankylosing spondylitis through simple immunological cross-reactivity or through interaction with HLA-B27 is unlikely.

The c-Jun and c-fos proto-oncogenes encode proteins that form a complex which regulates transcription from promoters containing AP-1 activation elements. c-Jun has specific DNA binding activity, while c-Fos has homology to the putative DNA binding domain of c-Jun. Following in vitro translation, c-Jun binds as a homodimer to the AP-1 DNA site, while c-Fos fails to dimerize and displays no apparent affinity for the AP-1 element. Cotranslated c-Jun and c-Fos proteins bind 25 times more efficiently to the AP-1 DNA site as a heterodimer than does the c-Jun homodimer. These experiments suggest that in growth factor-stimulated cells c-Jun binds DNA as a dimer with c-Fos as its natural partner. However, overexpression of c-Jun protein in the absence of c-Fos may result in formation of aberrant homodimeric transcription complexes, which could abrogate the normal mechanisms controlling gene expression.