Ikaros Null Mice Research Articles

Ikaros regulates B-cell tolerance

Abstract B-cell development and activation are tightly regulated at multiple steps to ensure a protective immune response, and at the same time avoiding harmful self-reactivity and autoimmunity. Ikaros is a transcription factor that is critical for B-cell development, as demonstrated by the complete lack of B-cells in Ikaros-null mice. Furthermore, Ikaros is shown to play important roles also at later stages of B-cell development, but the precise roles of Ikaros at different stages of B-cell development is still not fully understood. In recent years, Ikaros (encoded by the IKZF1 gene) has been linked to autoimmune disease in humans through both genome-wide association studies (GWAS) as well as recent reports of germline IKZF1 mutations in patients with autoimmune disease. However, the mechanisms underlying altered Ikaros function and the development of autoimmunity are not known. We previously developed Ikaros-mutant mouse models with targeted deletions of the exons encoding the DNA-binding zinc finger 1 (ZnF1) or ZnF4, and found that both mutants have B cells, but display selective partial defects at different stages of B-cell development. We recently found that the Ikzf1-ZnF4-mutant strain displays very high levels of serum Anti-Nuclear Antibodies (ANA) at young age, a hallmark of autoimmune disease. This indicates that Ikaros (and specifically exon 6 encoding ZnF4) is required to regulate B-cell tolerance, and we hypothesize that Ikaros regulates this, at least in part, by establishing a chromatin structure that sets restrictions on B-cell responses to limit autoreactive B cells. We are using our Ikaros-mutant mice to further study the role of Ikaros in B-cell development and tolerance, and will present our results to-date at the meeting.

Ikaros and its interacting partner CtBP target the metalloprotease ADAMTS10 to modulate pituitary cell function

We have previously described the expression and up-regulation of the C-terminal Binding Protein (CtBP) in response to pituitary hypoxia. This co-repressor interacts with the hematopoietic factor Ikaros to target several components implicated in cellular growth and apoptotic pathways. To identify common transcriptional pituitary targets we performed promoter arrays using Ikaros and CtBP chromatin immunoprecipitated (ChIP) DNA from pituitary AtT20 cells. This approach yielded a finite list of gene targets common to both transcription factors. Of these, the metalloprotease ADAMTS10 emerged as a validated target. We show the ability of Ikaros to bind the ADAMTS10 promoter, influence its transfected activity, and induce endogenous gene expression. ADAMTS10 is expressed in primary pituitary cells and is down-regulated in Ikaros null mice. Further, knockdown of ADAMTS10 in AtT20 cells recapitulates the impact of Ikaros deficiency on POMC/ACTH hormone expression. These results uncover a novel role for the metalloprotease ADAMTS10 in the pituitary. Additionally, they position this metalloprotease as a potential functional integrator of the Ikaros-CtBP chromatin remodeling network.

Regulation of gene expression dynamics during developmental transitions by the Ikaros transcription factor.

The DNA-binding protein Ikaros is a potent tumor suppressor and hematopoietic regulator. However, the mechanisms by which Ikaros functions remain poorly understood, due in part to its atypical DNA-binding properties and partnership with the poorly understood Mi-2/NuRD complex. In this study, we analyzed five sequential stages of thymocyte development in a mouse strain containing a targeted deletion of Ikaros zinc finger 4, which exhibits a select subset of abnormalities observed in Ikaros-null mice. By examining thymopoiesis in vivo and in vitro, diverse abnormalities were observed at each developmental stage. RNA sequencing revealed that each stage is characterized by the misregulation of a limited number of genes, with a strong preference for stage-specific rather than lineage-specific genes. Strikingly, individual genes rarely exhibited Ikaros dependence at all stages. Instead, a consistent feature of the aberrantly expressed genes was a reduced magnitude of expression level change during developmental transitions. These results, combined with analyses of the interplay between Ikaros loss of function and Notch signaling, suggest that Ikaros may not be a conventional activator or repressor of defined sets of genes. Instead, a primary function may be to sharpen the dynamic range of gene expression changes during developmental transitions via atypical molecular mechanisms that remain undefined.

Selective regulation of lymphopoiesis and leukemogenesis by individual zinc fingers of Ikaros

C2H2 zinc fingers are found in several transcriptional regulators in the immune system. However, these proteins usually contain more fingers than are needed for stable DNA binding, suggesting that different fingers regulate different genes and functions. Mice lacking finger 1 or finger 4 of Ikaros exhibited distinct subsets of the phenotypes of Ikaros-null mice. Most notably, the two fingers controlled different stages of lymphopoiesis and finger 4 was selectively required for tumor suppression. The distinct phenotypes suggest that only a small number of Ikaros target genes are critical for each of its biological functions. Subdivision of phenotypes and targets by mutagenesis of individual fingers will facilitate efforts to understand how members of this prevalent family regulate development, immunity and disease.

Role of Ikaros in hematopoiesis and tumor suppression: Selective functions of individual zinc fingers within the DNA-binding domain of Ikaros. (42.3)

Abstract Ikaros, a C2H2 zinc finger transcription factor, is a critical regulator of hematopoiesis and tumor suppression in the lymphoid lineage. The C2H2 zinc finger is the most prevalent DNA-binding motif in mammals, with DNA-binding domains usually containing more tandem fingers than are needed for stable sequence-specific DNA recognition. To examine the reason for the frequent presence of multiple zinc fingers, and to investigate in greater depth the role of Ikaros in hematopoiesis and tumor suppression, we generated mice lacking finger 1 or finger 4 of the 4-finger DNA-binding domain of Ikaros. Each mutant strain exhibited a specific subset of the phenotypes observed with Ikaros null mice. Of particular relevance, fingers 1 and 4 contributed to distinct stages of B- and T-cell development and finger 4 was selectively required for tumor suppression in thymocytes and in a new model of BCR-ABL+ acute lymphoblastic leukemia. These results, combined with transcriptome profiling and DNA-binding analysis, reveal that different subsets of fingers within multi-finger transcription factors can modulate binding to different target sequences and regulate distinct target genes and biological functions. These novel mutant strains provide a powerful tool to elucidate Ikaros' role in hematopoiesis and tumor suppression. Furthermore, this study demonstrates that selective mutagenesis can facilitate efforts to elucidate the functions and mechanisms of action of this prevalent class of factors.

Ikaros Is a GATA Switch Target Gene That Restrains Megakaryopoiesis In Part by Blocking Notch Signaling.

AbstractAbstract 1602The hematopoietic transcription factor Ikaros regulates the development of lymphoid cells. In particular, Ikaros has been shown to suppress expression of Notch target genes in developing thymocytes. In addition, Ikaros-deficient animals fail to develop B cells and instead display a T cell lymphoproliferative disorder. Furthermore, alterations in Ikaros gene expression are associated with human lymphoid neoplasms and loss of Ikaros is associated with progression of myeloproliferative neoplasms to acute myeloid leukemia. However, the role of Ikaros in normal and malignant myelopoiesis is not well characterized. Therefore, we first analyzed myeloid development in Ikaros deficient mice and observed extramedullary hematopoiesis in the spleen and a striking thrombocytosis in the peripheral blood of 8–9 week-old Ikaros-null mice (2×106 platelets/μl, compared to 0.5×106 platelets/μl in wild-type littermates). Flow cytometry, histology and colony forming assays revealed that the bone marrow and spleen of young Ikaros-null mice harbored a substantial increase in the numbers of megakaryocytes and myeloid cells. We next investigated how Ikaros activity is reduced during terminal differentiation. Previous reports have shown that GATA1-deficient and GATA1s mutant megakaryocytes, which are associated with Down syndrome acute megakaryoblastic leukemia (DS-AMKL), express aberrantly high levels of Ikaros, suggesting that Ikaros is a target of GATA-1 repression during terminal megakaryocyte differentiation. By chromatin immunoprecipitation assays, we found multiple sites in the Ikaros locus that are bound by GATA-2 in proliferating progenitors and by GATA-1 in maturing megakaryocytes. Furthermore, we discovered that GATA-1s fails to occupy these conserved Ikaros loci. Together these results strongly suggest that GATA-1, but not GATA-1s, displaces GATA-2 from the Ikaros gene during differentiation and that this GATA switch leads to repression of Ikaros, in turn allowing for terminal maturation of megakaryocytes. Since Notch participates in specification of the megakaryocyte lineage, we then asked whether Ikaros inhibited megakaryopoiesis by interfering with Notch signaling. Retroviral transduction of full-length Ikaros in murine common myeloid progenitors, but not in LSK cells, reduced megakaryocyte development upon Notch stimulation in OP9-DL1 stromal co-cultures. Ectopic expression of Ikaros in the 6133 cell line, which models the t(1;22) subtype of AMKL and shows aberrant Notch target genes activation by the OTT-MAL fusion oncogene, inhibited proliferation and induced apoptosis. Genome wide expression profiling in transduced 6133 cells confirmed that Ikaros alters expression of several genes involved in the Notch and growth factor signaling pathways. These results indicate that Ikaros restricts megakaryocyte development and inhibits proliferation of OTT-MAL-transformed AMKL cells at least in part by suppressing Notch signaling. Together, our results suggest that a functional antagonism between the Notch pathway and Ikaros controls normal megakaryocyte development and that this axis is deregulated in AMKL, contributing to aberrant expansion of immature megakaryoblasts. Disclosures:No relevant conflicts of interest to declare.

Notch Target Gene Deregulation and Maintenance of the Leukemogenic Phenotype Do Not Require RBP-Jκ in Ikaros Null Mice

Ikaros and Notch are transcriptional regulators essential for normal T cell development. Aberrant activation of Notch target genes is observed in Ikaros-deficient thymocytes as well as leukemia cell lines. However, it is not known whether Notch deregulation plays a preferential or obligatory role in the leukemia that arise in Ikaros null (Ik(-/-)) mice. To answer this question, the expression of the DNA-binding Notch target gene activator RBP-Jkappa was abrogated in Ik(-/-) double-positive thymocytes. This was accomplished through conditional inactivation using CD4-Cre transgenic mice containing floxed RBP-Jkappa alleles (RBPJ(fl/fl)). Ik(-/-) x RBPJ(fl/fl) x CD4-Cre(+) transgenic mice develop clonal T cell populations in the thymus that escape to the periphery, with similar kinetics and penetrance as their CD4-Cre(-) counterparts. The clonal populations do not display increased RBP-Jkappa expression compared with nontransformed thymocytes, suggesting there is no selection for clones that have not fully deleted RBP-Jkappa. However, RBPJ-deficient clonal populations do not expand as aggressively as their RBPJ-sufficient counterparts, suggesting a qualitative role for deregulated Notch target gene activation in the leukemogenic process. Finally, these studies show that RBP-Jkappa plays no role in Notch target gene repression in double-positive thymocytes but rather that it is Ikaros that is required for the repression of these genes at this critical stage of T cell development.

Ikaros null mice demonstrate defects in dendritic cell development. (36.17)

Abstract Ikaros is a transcription factor that is expressed almost exclusively in cells of the hematopoietic lineage. Ikaros null mice lack several hematopoietic cell lineages including some dendritic cell (DC) subsets. Additionally, several Ikaros null bone marrow (BM) progenitor populations have a decreased expression of the receptor tyrosine kinase, Flt3. DCs can be generated in vitro by culture of BM with either GM-CSF or Flt3L (the ligand for Flt3). Our studies find that loss of Ikaros leads to a specific defect in the ability of BM precursors to generate DCs in response to culture with Flt3L but not in response to culture with GM-CSF. Those DCs that do develop from the culture of Ikaros null BM with Flt3L have an activated phenotype with higher expression of MHC class II and the costimulatory molecules CD40, CD80 and CD86 than their wild type counterparts. An examination of gene expression in Ikaros null BM progenitors cultured with Flt3L shows an inappropriately low expression of Irf4, Irf8 and Spib, genes encoding transcription factors known to be important for DC development. Studies are currently underway to determine whether enforced expression of Ikaros, Flt3 or one of the above-mentioned transcription factors can rescue DC development upon culture of Ikaros null BM with Flt3L. These studies will provide a potential molecular mechanism for the impaired development of DCs in Ikaros null mice.

Cutting Edge: Ikaros Is a Regulator of Th2 Cell Differentiation

Ikaros, a hematopoietic transcription factor, has well defined effects on early lymphocyte development in the bone marrow and thymus. In this study we demonstrate that Ikaros is a positive regulator of Th2 cytokine gene expression in peripheral T cells. CD4+ T cells from naive Ikaros(null) mice cultured under Th2-skewing conditions express the Th1 cytokine IFN-gamma and have reduced IL-4, IL-5, and IL-13 expression. Ikaros directly associates with several Th2 locus regulatory regions in naive CD4+ T cells. The decreased ability to express Th2 cytokines in Ikaros(null)T cells corresponds with histone 3 hypoacetylation across the Th2 cytokine locus as well as decreased GATA3 and cMaf and increased T-bet and STAT1 expression. These data support a model whereby Ikaros directly activates Th2 gene expression by promoting local chromatin accessibility during CD4+ T cell differentiation and also acts indirectly to regulate expression of Th2- and Th1-specific transcription factors.

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.

An essential role for the hematopoietic transcription factor Ikaros in hypothalamic–pituitary-mediated somatic growth

Ikaros transcription factors play critical functions in the control of lymphohematopoiesis and immune regulation. Family members contain multiple zinc fingers that mediate DNA binding and homooligomerization or heterooligomerization. Ikaros is abundantly expressed in pituitary mammosomatotrophs, where it deacetylates histone 3 sites on the proximal growth hormone (GH) promoter to silence gene expression. Ikaros-null mice display stunted growth with reduced circulating levels of the GH target factor insulin-like growth factor I (IGF-I). Ikaros-deficient mice have small anterior pituitary glands with a disproportionately reduced somatotroph population. Systemic administration of GH results in increased IGF-I levels and enhanced somatic growth. In contrast, reconstitution with WT lymphocytes was not sufficient to rescue the stunted growth phenotype of Ikaros-deficient mice. Ikaros was identified in mouse hypothalamic arcuate nuclei, where it colocalized with GH-releasing hormone (GHRH); in contrast, Ikaros-null mice lack GHRH immunoreactivity in the hypothalamus. Overexpression of Ikaros enhanced GHRH promoter activity and induced endogenous GHRH gene expression. These findings unmask a wider role for Ikaros in the neuroendocrine system, highlighting a critical contribution to the development of the hypothalamic-pituitary somatotrophic axis.

Ikaros increases normal apoptosis in adult erythroid cells

Ikaros is a critical transcriptional regulator of hematopoietic cell differentiation. In addition to its effects on the lymphoid system and hematopoietic stem-cell compartment, we have previously shown that Ikaros is also required for normal erythroid development. In this report, we compare Ikaros-dependent gene expression in erythroid cells of mice lacking the Ikaros protein with that of normal mice in purified adult bone-marrow erythroid cells (BMRC). Gene expression, measured by Affymetrix microarray analysis, indicates that in the BMRC of Ikaros-null mice, there is significant up-regulation of SMADs 6 and 7, serine protease inhibitor 3, and immediate-early protein 3 (IER3), all proteins that play a modulating role in apoptosis. We investigate the role of Ikaros in oxidative stress-induced apoptosis using Annexin-V staining and FACS analysis. We find a decrease in apoptosis in the BMRC of Ikaros-null mice compared to normal mice. This effect is also seen in nonerythroid cells but is stronger in BMRC. We conclude that normal Ikaros function increases normal apoptosis in erythroid cells. The data also suggest that Ikaros plays a role in apoptosis-mediated events in other normal hematopoietic cell lineages.

Ikaros Induces Quiescence and T-Cell Differentiation in a Leukemia Cell Line

Ikaros is a hematopoietic cell-specific zinc finger DNA binding protein that plays an important role in lymphocyte development. Genetic disruption of Ikaros results in T-cell transformation. Ikaros null mice develop leukemia with 100% penetrance. It has been hypothesized that Ikaros controls gene expression through its association with chromatin remodeling complexes. The development of leukemia in Ikaros null mice suggests that Ikaros has the characteristics of a tumor suppressor gene. In this report, we show that the introduction of Ikaros into an established mouse Ikaros null T leukemia cell line leads to growth arrest at the G0/G1 stage of the cell cycle. This arrest is associated with up-regulation of the cell cycle-dependent kinase inhibitor p27kip1, the induction of expression of T-cell differentiation markers, and a global and specific increase in histone H3 acetylation status. These studies provide strong evidence that Ikaros possesses the properties of a bona fide tumor suppressor gene for the T-cell lineage and offer insight into the mechanism of Ikaros's tumor suppressive activity.

Multiple hematopoietic defects and delayed globin switching in Ikaros null mice.

We have previously reported the structure of a chromatin remodeling complex (PYR complex) with Ikaros as its DNA binding subunit that is specifically present in adult murine and human hematopoietic cells. We now show that homozygous Ikaros "knockout" (null) mice lack the PYR complex, demonstrating the requirement for Ikaros in the formation of the complex on DNA. Heterozygous Ikaros null mice have about half as much PYR complex, indicating a dosage effect for both Ikaros and PYR complex. We also show that Ikaros null mice have multiple hematopoietic cell defects including anemia and megakaryocytic abnormalities, in addition to previously reported lymphoid and stem cell defects. The null mice also have a delay in murine embryonic to adult beta-globin switching and a delay in human gamma to beta switching, consistent with a previously suggested role for PYR complex in this process. Lastly, cDNA array analyses indicate that several hematopoietic cell-specific genes in all blood lineages are either up- or down-regulated in 14-day embryos from Ikaros null as compared with wild-type mice. These results indicate that Ikaros and PYR complex function together in vivo at many adult hematopoietic cell-specific genes and at intergenic sites, affecting their expression and leading to pleiotropic hematopoietic defects.

Helios, a novel dimerization partner of Ikaros expressed in the earliest hematopoietic progenitors.

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. 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. 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.

Selective Defects in the Development of the Fetal and Adult Lymphoid System in Mice with an Ikaros Null Mutation

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 γδ 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.

Transcription Factors and Hematopoietic Development

Transcription Factors and Hematopoietic Development