Absence Of Ikaros Research Articles

KCTD5 regulates Ikaros degradation induced by chemotherapeutic drug etoposide in hematological cells.

Therapy-related leukemia carries a poor prognosis, and leukemia after chemotherapy is a growing risk in clinic, whose mechanism is still not well understood. Ikaros transcription factor is an important regulator in hematopoietic cells development and differentiation. In the absence of Ikaros, lymphoid cell differentiation is blocked at an extremely early stage, and myeloid cell differentiation is also significantly affected. In this work, we showed that chemotherapeutic drug etoposide reduced the protein levels of several isoforms of Ikaros including IK1, IK2 and IK4, but not IK6 or IK7, by accelerating protein degradation, in leukemic cells. To investigate the molecular mechanism of Ikaros degradation induced by etoposide, immunoprecipitation coupled with LC-MS/MS analysis was conducted to identify changes in protein interaction with Ikaros before and after etoposide treatment, which uncovered KCTD5 protein. Our further study demonstrates that KCTD5 is the key stabilizing factor of Ikaros and chemotherapeutic drug etoposide induces Ikaros protein degradation through decreasing the interaction of Ikaros with KCTD5. These results suggest that etoposide may induce leukemic transformation by downregulating Ikaros via KCTD5, and our work may provide insights to attenuate the negative impact of chemotherapy on hematopoiesis.

Cross Talk between Ikaros and RUNX1 in Myeloid Leukemia

Background: RUNX1 is critically important for normal hematopoiesis within both the lymphoid and myeloid lineages. Mutations or translocations involving RUNX1 have been identified in a variety of myeloid and lymphoid lineage hematopoietic malignancies. RUNX1 interacts with chromatin remodeling complexes and modulates transcriptional regulation of target genes via epigenetic mechanisms.IKAROS is a transcription factor that plays an essential role in normal lymphopoiesis, and mutations of IKZF1, the gene encoding IKAROS, are frequently seen in acute lymphoblastic leukemia (ALL). Despite reports of the presence of IKZF1 mutations in acute myeloid leukemia (AML), there remains a paucity of information regarding a possible role for disrupted IKAROS function in AML.Emerging evidence indicates that the transcriptional programs of these two transcription factors converge on critical pathways in hematopoiesis, such as the Notch1 signaling pathway. We are testing the hypothesis that IKAROS not only contributes to a gene regulatory program in AML, but specifically that the IKAROS and RUNX1 regulatory networks intersect in the pathogenesis of myeloid malignancies.Methods: Chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) was utilized to identify the genome-wide binding sites of IKAROS and RUNX1 in the human myeloid sarcoma U937 cell-line. Independent ChIP-seq replicates were generated for these transcription factors, as well as replicates for a panel of activating and repressive histone modifications. The genes bound by IKAROS and RUNX1 as well as the chromatin patterns of these binding sites were analyzed with respect to their pathways were analyzed to understand the mutual and shared roles of these transcription factors.Results: We identified that greater than 30% of RUNX1 binding sites in this myeloid sarcoma cell line are also bound by IKAROS. These sites of co-occupancy are enriched at distal sites marked by the enhancer histone mark H3K27ac. Of the high-confidence IKAROS-RUNX1 shared binding sites, RUNX1 binding motifs predominate; while ETS, IRF4 and CEBPA motifs are also present. A Genome Regions Enrichment of Annotations (GREAT) analysis of the high confidence IKAROS-RUNX1 shared binding sites reveals enrichment for several biological associations, including leukocyte activation, migration, and chemotaxis. Importantly, genes associated with the regulation of chromatin organization and Notch and NFKB signaling cascades are bound by these factors.We have additionally found that IKAROS and RUNX1 are bound to the RUNX1 gene promoter. IKAROS is also independently bound to the RUNX1 gene at an upstream enhancer, also marked by H3K27ac.Conclusions: The role of IKAROS as a tumor suppressor in lymphoid leukemias is well established. Here, we begin to examine a role for IKAROS in myeloid leukemogenesis by defining the IKAROS cistrome in a myeloid sarcoma cell line. We have additionally identified cross talk between IKAROS and the important pan-hematopoietic transcription factor RUNX1 in myeloid malignancy by showing bidirectional transcriptional regulation between these two tumor suppressors, and defining an overlapping set of transcriptionally regulated genes. Future studies, including RNA-Seq experiments in the presence and absence of IKAROS and RUNX1 knockdown, will define the transcriptome, explore the functional consequences of co-occupancy at these identified binding sites, and functionally characterize the relationship between IKAROS and RUNX1 in the context of acute myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Deregulation of Ikaros expression in B-1 cells: New insights in the malignant transformation to chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a chronic form of leukemia that originates from an abnormal expansion of CD5+ B-1 cells. Deregulation in the BCR signaling is associated with B-cell transformation. Contrariwise to B-2 cells, BCR engagement in B-1 cells results in low proliferation rate and increased apoptosis population, whereas overactivation may be associated with lymphoproliferative disorders. It has been demonstrated that several transcription factors that are involved in the B cell development play a role in the regulation of BCR function. Among them, Ikaros is considered an essential regulator of lymphoid differentiation and activation. Several reports suggest that Ikaros expression is deregulated in different forms of leukemia. Herein, we demonstrated that CLL cells show decreased Ikaros expression and abnormal cytoplasmic cell localization. These alterations were also observed in radioresistant B-1 cells, which present high proliferative activity, suggesting that abnormal localization of Ikaros could determine its loss of function. Furthermore, Ikaros knockdown increased the expression of BCR pathway components in murine B-1 cells, such as Lyn, Blnk, and CD19. Additionally, in the absence of Ikaros, B-1 cells become responsive to BCR stimulus, increasing cell proliferation even in the absence of antigen stimulation. These results suggested that Ikaros is an important controller of B-1 cell proliferation by interfering with the BCR activity. Therefore, altered Ikaros expression in CLL or radioresistant B-1 cells could determine a responsive status of BCR to self-antigens, which would culminate in the clonal expansion of B-1 cells.

Lack of Ikaros Deregulates Inflammatory Gene Programs in T Cells.

CD4 Th cells are organizers of the immune response, directing other immune cells to initiate and maintain effective humoral and cellular immunity. CD4 T cells differentiate into distinct Th effector or regulatory subsets in response to signals delivered to them during the course of infection. Ikaros is a transcription factor that is expressed in blood cells from the level of the hematopoietic stem cell. It is required for normal thymic T cell development and serves as a tumor suppressor, as lack of Ikaros in developing lymphoid cells results in leukemia. To study the role of Ikaros in CD4 T cell differentiation and function, an Ikaros conditional knockout mouse was developed such that Ikaros expression was deleted specifically in mature T cells, thus avoiding defects observed in germline Ikaros mutant mice. Using this model system, we have shown that in the absence of Ikaros, CD4 T cells are able to attain Th1, Th2, and Th17, but not inducible regulatory T, cell fates. However, they show enhanced expression of a cohort of proinflammatory cytokines, resulting in differentiation of Th17 cells with a phenotype that has been associated with autoimmunity and pathological inflammation. In addition, we define Ikaros as a repressor of the gene program associated with the response to type I IFNs, another key pathway whose deregulation is linked to autoimmunity. Taken together, these data definitively define Ikaros as a critical regulator at the center of the inflammatory response in T cells and highlight a potential role in suppressing autoimmunity.

Dual Roles for Ikaros in Regulation of Macrophage Chromatin State and Inflammatory Gene Expression

Macrophage activation by bacterial LPS leads to induction of a complex inflammatory gene program dependent on numerous transcription factor families. The transcription factor Ikaros has been shown to play a critical role in lymphoid cell development and differentiation; however, its function in myeloid cells and innate immune responses is less appreciated. Using comprehensive genomic analysis of Ikaros-dependent transcription, DNA binding, and chromatin accessibility, we describe unexpected dual repressor and activator functions for Ikaros in the LPS response of murine macrophages. Consistent with the described function of Ikaros as transcriptional repressor, Ikzf1-/- macrophages showed enhanced induction for select responses. In contrast, we observed a dramatic defect in expression of many delayed LPS response genes, and chromatin immunoprecipitation sequencing analyses support a key role for Ikaros in sustained NF-κB chromatin binding. Decreased Ikaros expression in Ikzf1+/- mice and human cells dampens these Ikaros-enhanced inflammatory responses, highlighting the importance of quantitative control of Ikaros protein level for its activator function. In the absence of Ikaros, a constitutively open chromatin state was coincident with dysregulation of LPS-induced chromatin remodeling, gene expression, and cytokine responses. Together, our data suggest a central role for Ikaros in coordinating the complex macrophage transcriptional program in response to pathogen challenge.

Ikaros deficiency in host hematopoietic cells separates GVL from GVHD after experimental allogeneic hematopoietic cell transplantation

The graft-versus-leukemia (GVL) effect following allogeneic hematopoietic stem cell transplantation (allo-HCT) is critical for its curative potential. Hwever, GVL is tightly linked to graft-versus-host disease (GVHD). Among hematological malignancies, acute lymphoblastic leukemia (ALL) is the most resistant to GVL, although the reasons for this remain poorly understood. Clinical studies have identified alterations in Ikaros (Ik) transcription factor as the major marker associated with poor outcomes in ALL. We have shown that the absence of Ik in professional host-derived hematopoietic antigen-presenting cells (APCs) exacerbates GVHD. However, whether Ik expression plays a role in resistance to GVL is not known. In this study we used multiple clinically relevant murine models of allo-HCT to explore whether Ik expression in hematopoietic APCs and/or leukemic cells is critical for increasing resistance to GVL and thus inducing relapse. We found that Ik deficiency in host APCs failed to enhance GVL despite increased GVHD severity. Mechanistic studies with bone marrow (BM) chimeras and tetramer analyses demonstrated reduced tumor-specific immunodominant (gag+) antigen responses in the [B6Ik−/−→B6] group. Loss of GVL was observed when both the leukemia cells and the host APCs were deficient in Ik. We found that calreticulin (CRT) expression in host antigen-presenting dendritic cells (DCs) of Ik−/− animals was significantly lower than in wild-type animals. Rescuing CRT expression in Ik−/− DCs improved leukemic-specific cytotoxic T cell function. Together, our data demonstrate that the absence of Ikaros in host hematopoietic cells promotes resistance to GVL despite increasing GVHD and thus provides a potential mechanism for the poor outcome of Ik−/− ALL patients.

Defective Flt3L driven DC development in the absence of Ikaros (111.35)

Abstract Ikaros null (Ik-) mice have impaired dendritic cell (DC) development. This defect is recapitulated when Ik- bone marrow (BM) is cultured with Flt3L, a DC growth factor. Ik- BM cultures generate significantly fewer DCs than their wild type (WT) counterparts. This phenotype is specific to Flt3L-driven DC developments as Ik- GM-CSF cultures generate normal numbers of DCs. Time course gene expression analyses of Flt3L-cultures show that genes important for DC development such as Irf4, Irf8 and Spib are variably under-expressed in both Ik- progenitors and mature DCs, with Spib being the most affected. These data demonstrate that low expression of DC genes is not solely due to decreased DC differentiation and suggest that Ikaros may regulate their expression, which we are now exploring. The DCs that develop in Ik- Flt3L cultures are also qualitatively different from their WT counterparts. They have an activated phenotype, implicating Ikaros in regulation of DC function as well. Enforced expression of full-length Ikaros (Ik1) in Ik- BM cannot restore Flt3L-driven DC development, suggesting a different Ikaros isoform may be needed for DC rescue. Studies now underway will define Ikaros isoform expression during DC development and determine if enforced expression of discrete Ikaros isoforms or the Flt3 receptor itself can restore Flt3L-driven DC development in Ik- BM. These studies will place Ikaros relative to Flt3 signals during DC development and define its molecular role.

FoxO1 induces Ikaros splicing to promote immunoglobulin gene recombination

Somatic rearrangement of immunoglobulin (Ig) genes is a key step during B cell development. Using pro-B cells lacking the phosphatase Pten (phosphatase and tensin homolog), which negatively regulates phosphoinositide-3-kinase (PI3K) signaling, we show that PI3K signaling inhibits Ig gene rearrangement by suppressing the expression of the transcription factor Ikaros. Further analysis revealed that the transcription factor FoxO1 is crucial for Ikaros expression and that PI3K-mediated down-regulation of FoxO1 suppresses Ikaros expression. Interestingly, FoxO1 did not influence Ikaros transcription; instead, FoxO1 is essential for proper Ikaros mRNA splicing, as FoxO1-deficient cells contain aberrantly processed Ikaros transcripts. Moreover, FoxO1-induced Ikaros expression was sufficient only for proximal V(H) to DJ(H) gene rearrangement. Simultaneous expression of the transcription factor Pax5 was needed for the activation of distal V(H) genes; however, Pax5 did not induce any Ig gene rearrangement in the absence of Ikaros. Together, our results suggest that ordered Ig gene rearrangement is regulated by distinct activities of Ikaros, which mediates proximal V(H) to DJ(H) gene rearrangement downstream of FoxO1 and cooperates with Pax5 to activate the rearrangement of distal V(H) genes.

Concerted action of Helios and Ikaros controls the expression of the inositol 5‐phosphatase SHIP

Ikaros family transcription factors have a key role in lymphoid development, and their aberrant function contributes to a multitude of lymphoid malignancies. Ikaros and Helios bind to similar DNA sequences, and Helios associates with Ikaros-containing chromatin remodeling complexes. Previously, we have shown that loss of Ikaros leads to diminished BCR-signaling strength. In this study, we describe a Helios-deficient chicken DT40 B-cell line with a BCR signaling phenotype that is the opposite to that of Ikaros-deficient cells. In contrast to Ikaros-deficient cells, Helios(-/-) B cells exhibit increased calcium release to the cytoplasm after BCR crosslinking, but diminished BCR-induced phosphorylation of signaling molecules. The inositol 5-phosphatase SHIP, an important regulator in several signaling pathways, is differentially expressed in Ikaros- and Helios-deficient cells. In the absence of Ikaros, SHIP is upregulated, whereas Helios deficiency leads to the downregulation of SHIP expression. We also show with ChIP that Ikaros binds to the promoter of the INPP5D gene-encoding SHIP. Considering the critical role of SHIP in the BCR signaling pathway, our findings provide insight into the mechanism of how both Helios and Ikaros are involved in the regulation of BCR signaling.

Ikaros Deficiency Enhances Notch Signaling in Host Antigen Presenting Cells and Differentially Regulates Gvhd and GVL Responses After Experimental BMT.

Abstract 690Host antigen presenting cells (APCs) are necessary for induction of graft-versus-host (GVH) responses. But the APC autonomous molecular mechanisms that are critical for modulation of GVH are not known. Ikaros (Ik) is a zinc-finger DNA binding protein that is essential for the development of lymphoid cells and hematological malignancies. Because Ik is known to negatively regulate certain dendritic cell (DC), the most potent APCs, functions (Wu et al, Immunity, 1997) we hypothesized that its deficiency in host APCs will reduce GVHD. We generated [B6→B6] and [Ik-/-B6→B6] chimeras and utilized them as recipients in well characterized MHC matched, minor mismatched C3H.SW B6 model of acute GVHD. [B6→B6] and [Ik/-B6 → B6] animals received 9 Gy and were transplanted with 3 × 106 T cells and 5 × 106 BM from either syngeneic B6 or allogeneic C3H.SW donors. Contrary to our hypothesis, [Ik-/-B6 < 0.001). The increase in mortality was associated with a significant increase in GVHD specific clinical severity, histopathological damage of the liver and GI tract, donor T cell expansion, serum levels TNF-αa, IFNγ and IL-17 on days +7 and +14 after BMT. Similar results were obtained in a second, BALB/c→B6 model of GVHD and also in recipient chimeras generated from dominant negative Ikaros (IkDN) mice, demonstrating a role for Ik in a strain independent manner. We next performed systematic analyses of DCs to uncover the mechanisms underpinning our surprising results. Phenotypic analysis of spleen and BM derived DCs demonstrated similar numbers of CD8+ and CD8-CD11c+DCs but a reduced number of pDCs were present in Ik-/- animals when compared with WT animals. Ik-/-DCs demonstrated an immature phenotype as determined by the expression of CD40/80. By contrast, functional analyses of Ik-/- DCs, when stimulated with LPS showed significantly greater secretion of proinflammatory cytokines TNF-αa and IL-6 and greater stimulation of allo-T cells in MLRs when compared with WT B6 DCs. CFSE, annexin and CD107a labeling studies demonstrated that Ik-/-DCs caused greater proliferation without altering the rate of apoptosis or the cytotoxic function of the allo-T cells. To characterize the molecular mechanisms we evaluated the role of putative molecular targets of Ikaros, the Notch signaling pathway because Ikaros and Notch antagonistically regulate target genes (Chari et al. J Immunol 2008 and Bugeon, et al. J Immunol 2008). Both Ik-/- and IkDN DCs, at steady state, showed an increase in the expression of several Notch target genes such as Hes-1, Hex-1, Deltex1, Notch3, Jagged-1 and Jagged-2. Blockade of Notch signaling with γ-secretase inhibitor (DAPT) in MLR mitigated the enhanced allo-stimulatory capacity of the Ik-/- DCs, thus demonstrating a functional relevance for enhanced notch signaling in the absence of Ikaros. We next hypothesized that given the enhanced GVHD response that was associated with increased proliferation and preserved cytotoxicity of allo-T cells, the GVL response will also be enhanced in the recipients with Ik-/- APCs. Unexpectedly, the [B6→ B6] and [Ik-/-B6 → B6] chimeras when transplanted with allogeneic T cells from C3H.SW cells along with two different doses of syngeneic 0.5 or 1×104 MBL-2 tumor cells demonstrated equivalent GVL responses despite greater severity of GVHD. Together our data demonstrate (a) for the first time, to our knowledge, suggest differential regulation of GVHD and GVL at the level of host APCs and (b) show a role for a novel molecular pathway, the Ik-Notch axis, in the host APCs as an important modulator of GVH responses. Disclosures:Maillard:University of Michigan Comprehensive Cancer Center: Research Funding; Damon Runyon Cancer Foundation: Research Funding; ASH Scholar Awards : Research Funding.

Notch target gene deregulation and leukemogenesis occur independently of RBPJ in Ikaros null thymocytes. (138.23)

Abstract Both Ikaros and Notch are transcriptional regulators that are essential for normal T cell development. Cooperative mutations causing a reduction in Ikaros activity and an increase in Notch activation have been hypothesized to promote T cell leukemia. However, it is not known whether Notch deregulation plays a preferential or obligatory role in the leukemias that arise in Ikaros null (Ik-/-) mice with 100% penetrance. In order to answer this question, we abrogated Notch target gene activation in Ik-/- thymocytes using conditional inactivation of the DNA-binding Notch-target gene activator RBP-J. Through analyses of these mice we have made two important discoveries. First, thymocytes that lack either Ikaros alone or both Ikaros and RBP-J exhibit high level derepression of Notch target genes. However, this derepression is not observed in thymocytes lacking RBP-J alone, which suggests that Ikaros, but not RBP-J, is required for Notch target gene repression in DP thymocytes. This data represents a major shift in our understanding of regulatory factors involved in Notch target gene repression. Secondly, Ik-/- mice develop clonal T cell populations, a hallmark of leukemia, with similar kinetics in the presence or absence of RBP-J. The appearance of clonal populations in the thymus and spleen of Ik-/- mice lacking RBP-J indicates that T cell transformation in the absence of Ikaros does not require deregulated Notch signaling.

Cutting Edge: Ikaros Null Thymocytes Mature into the CD4 Lineage with Reduced TCR Signal: A Study Using CD3ζ Immunoreceptor Tyrosine-Based Activation Motif Transgenic Mice

Positive selection is a critical T cell developmental checkpoint that is driven by TCR signals. Enhanced positive selection toward the CD4 lineage occurs in the absence of Ikaros. One explanation for this phenotype is that Ikaros establishes the TCR signaling threshold that must be overcome for positive selection to occur. In the current study, this possibility is explored through the use of CD3zeta ITAM transgenic mice that express a CD3 zeta-chain with zero, one, or three ITAMs and an MHC class II (DO11.10)- or MHC class I (H-Y)-restricted TCR transgene. Using this system, we demonstrate that in the absence of Ikaros, thymocytes are able to mature into the CD4 lineage with reduced TCR signaling potential compared with that required to drive the maturation of wild-type thymocytes. We also demonstrate that maturation into the CD8 lineage is enhanced under conditions of reduced TCR signaling potential in the absence of Ikaros.

Ikaros and GATA-1 Combinatorial Effect Is Required for Silencing of Human γ-Globin Genes

During development and erythropoiesis, globin gene expression is finely modulated through an important network of transcription factors and chromatin modifying activities. In this report we provide in vivo evidence that endogenous Ikaros is recruited to the human beta-globin locus and targets the histone deacetylase HDAC1 and the chromatin remodeling protein Mi-2 to the human gamma-gene promoters, thereby contributing to gamma-globin gene silencing at the time of the gamma- to beta-globin gene transcriptional switch. We show for the first time that Ikaros interacts with GATA-1 and enhances the binding of the latter to different regulatory regions across the locus. Consistent with these results, we show that the combinatorial effect of Ikaros and GATA-1 impairs close proximity between the locus control region and the human gamma-globin genes. Since the absence of Ikaros also affects GATA-1 recruitment to GATA-2 promoter, we propose that the combinatorial effect of Ikaros and GATA-1 is not restricted to globin gene regulation.

Antagonistic Interactions between Ikaros and the Chromatin Remodeler Mi-2β Determine Silencer Activity and Cd4 Gene Expression

Lineage commitment is induced by changes in gene expression dictated by the intimate interaction between transcription factors and chromatin regulators. Here, we revealed the antagonistic interplay between Ikaros and its associate the chromatin remodeler Mi-2beta during T cell development, as exemplified by the regulation of Cd4 expression. Loss of Ikaros or Mi-2beta led to activation or repression, respectively, of the Cd4 locus at inappropriate stages of development. Their combined mutation reverted to normal CD4 expression. In double-negative thymocytes, Ikaros binding to the Cd4 silencer contributed to its repressive activity. In double-positive thymocytes, concomitant binding of Mi-2beta with Ikaros to the Cd4 silencer caused silencer inactivation, thereby allowing for CD4 expression. Mi-2beta facilitated recruitment of histone acetyl transferases to the silencer. This recruitment possibly antagonized Ikaros and associated repressive activities. Thus, concomitant interactions between functionally opposing chromatin-regulating machineries are an important mode of gene regulation during lineage determination.

Early hematopoietic lineage restrictions directed by Ikaros

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.

Ikaros has a crucial role in regulation of B cell receptor signaling

The transcription factor Ikaros, a key regulator of hematopoiesis, has an essential role in lymphocyte development. In mice, fetal lymphoid differentiation is blocked in the absence of Ikaros, and whereas T cells develop postnatally, B cells are totally absent. The significance of Ikaros in the B cell development is evident, but how Ikaros regulates B cell function has neither been established nor previously been studied with B cells that lack Ikaros expression. Here we show that disruption of Ikaros in the chicken B cell line DT40 induces a B cell receptor (BCR) signaling defect with reduced phospholipase Cgamma2 phosphorylation and impaired intracellular calcium mobilization, which is restored by Ikaros reintroduction. Furthermore, we show that lack of Ikaros induces hyperphosphorylation of Casitas B lymphoma protein subsequent to BCR activation. These results indicate that the absolute need of Ikaros for development, cell fate decisions and maintenance of B cells is due to the enhancement of BCR signaling.

Unconventional Potentiation of Gene Expression by Ikaros

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.

Defects in hemopoietic stem cell activity in Ikaros mutant mice.

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.

Pre–T Cell Receptor (Tcr) and Tcr-Controlled Checkpoints in T Cell Differentiation Are Set by 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.

Aiolos, a lymphoid restricted transcription factor that interacts with Ikaros to regulate lymphocyte differentiation.

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.