Katia Georgopoulos

The ikaros gene is required for the development of all lymphoid lineages

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.

c-Jun dimerizes with itself and with c-Fos, forming complexes of different DNA binding affinities

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.

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 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.

Ikaros DNA-Binding Proteins Direct Formation of Chromatin Remodeling Complexes in Lymphocytes

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.

A dominant mutation in the Ikaros gene leads to rapid development of leukemia and lymphoma

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 functionally diverse zinc finger DNA-binding proteins.

We previously described the lymphocyte-restricted Ikaros gene encoding a zinc finger DNA-binding protein as a potential regulator of lymphocyte commitment and differentiation. Here, we report the isolation of four additional Ikaros transcripts, products of alternate splicing that encode functionally diverse proteins. The Ikaros proteins contain unique combinations of zinc finger modules that dictate their overall sequence specificity and affinity. The Ik-1 and Ik-2 proteins can both bind, albeit with different affinities, to the same recognition sequences present in a number of lymphocyte-specific regulatory elements. The Ik-3 and the Ik-4 proteins interact only with a subset of these motifs. The Ik-1 and Ik-2 proteins can strongly stimulate transcription, whereas Ik-3 and Ik-4 are weak activators. Significantly, the transcription activation potential of the Ikaros proteins correlates with their subcellular localization. Upon ectopic expression of the Ikaros isoforms in nonlymphoid cells, Ik-1 and Ik-2 localize to the nucleus, whereas Ik-3 and Ik-4 are predominantly found in the cytoplasm. The Ikaros isoforms are expressed differentially in lymphocytes: Ik-1 and Ik-2 mRNAs are the predominating forms, and Ik-4 is present in significant amounts only in early T-cell progenitors, whereas Ik-3 and Ik-5 transcripts are expressed at relatively low levels throughout lymphocyte development. The ability of the Ikaros gene to generate functionally diverse proteins that may participate in distinct regulatory pathways substantiates its role as a master regulator during lymphocyte development.

Zinc finger-mediated protein interactions modulate Ikaros activity, a molecular control of lymphocyte development.

The Ikaros gene, an essential regulator of lymphocyte differentiation, encodes, by means of differential splicing, protein isoforms with a distinct number of Kruppel‐type zinc fingers organized in two domains. Deletion of the N‐terminal zinc finger domain responsible for the sequence‐specific DNA binding of the Ikaros proteins results in an early and complete arrest in lymphocyte development in homozygous mutant mice. In sharp contrast, heterozygotes reliably develop T cell leukemias and lymphomas. Here we show that the C‐terminal zinc finger domain present in all of the Ikaros wild‐type and mutant isoforms is responsible for their stable interactions off DNA and plays a pivotal role in determining their overall activity. Mutations in the C‐terminal zinc fingers which ablate Ikaros protein interactions have a dramatic effect on the ability of these proteins to bind DNA and activate transcription. Therefore, interactions between Ikaros isoforms with an intact DNA binding domain are essential for their function. In contrast, interactions between isoforms with and without a DNA binding domain result in Ikaros complexes that do not bind DNA and, as a consequence, cannot activate transcription. Dominant‐negative Ikaros isoforms are generated in smaller amounts by the wild‐type Ikaros gene but are also produced exclusively by the N‐terminally deleted Ikaros locus. Given these data, we propose that interactions between Ikaros isoforms are essential for normal progression through the lymphoid pathways. Mutations in the Ikaros gene that prevent Ikaros protein interactions or which change the relative ratio of DNA to non‐DNA binding isoforms have profound effects in both lymphoid specification and homeostasis.

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.

Haematopoietic cell-fate decisions, chromatin regulation and ikaros

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.

Repression by Ikaros and Aiolos is mediated through histone deacetylase complexes

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.