Derivation of dendritic cells from myeloid and lymphoid precursors.

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.

The follicular versus marginal zone B lymphocyte cell fate decision is regulated by Aiolos, Btk, and CD21.

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.

Identification and characterization of a transcriptional regulator for the lck proximal promoter.

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.

Ikaros interactions with CtBP reveal a repression mechanism that is independent of histone deacetylase activity.

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.

Distinct signals control the hematopoiesis of lymphoid-related dendritic 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)

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.

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.

Control of lymphocyte development by the Ikaros gene family.

Lymphoid cell differentiation relies on precisely orchestrated gene activation and repression events. Gene targeting studies have demonstrated crucial roles for the transcription factors Ikaros and Aiolos in regulating multiple stages of B and T cell development. Recent experiments suggest that Ikaros and Aiolos set B cell antigen-receptor (BCR)- and TCR-mediated signaling thresholds and that the molecules exist within T cells in nuclear complexes that contain nucleosome remodeling and histone deacetylase activities.

Ikaros sets thresholds for T cell activation and regulates chromosome propagation.

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.

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.

The linkage between T-cell and dendritic cell development in the mouse thymus.

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.

Aiolos regulates B cell activation and maturation to effector state.

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.

Ikaros in hemopoietic lineage determination and homeostasis.

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.

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

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.

Cell-autonomous defects in dendritic cell populations of Ikaros mutant mice point to a developmental relationship with the lymphoid lineage.

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.

Transcription factors required for lymphoid lineage commitment.

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.