Ikaros is essential for pre-BCR down-regulation, Igκ germline transcription, Ig light chain recombination, and pre-B cell differentiation, in part by antagonizing IL-7–dependent gene regulation.
Pre-B cell receptor (pre-BCR) signaling and migration from IL-7–rich environments cooperate to drive pre-B cell differentiation via transcriptional programs that remain unclear. We show that the Ikaros transcription factor is required for the differentiation of large pre-B to small pre-B cells. Mice deleted for Ikaros in pro/pre-B cells show a complete block of differentiation at the fraction C′ stage, and Ikaros-null pre-B cells cannot differentiate upon withdrawal of IL-7 in vitro. Restoration of Ikaros function rescues pre-B cell differentiation in vitro and in vivo and depends on DNA binding. Ikaros is required for the down-regulation of the pre-BCR, Igκ germline transcription, and Ig L chain recombination. Furthermore, Ikaros antagonizes the IL-7–dependent regulation of >3,000 genes, many of which are up- or down-regulated between fractions C′ and D. Affected genes include those important for survival, metabolism, B cell signaling, and function, as well as transcriptional regulators like Ebf1, Pax5, and the Foxo1 family. Our data thus identify Ikaros as a central regulator of IL-7 signaling and pre-B cell development.
Different methods have been proposed for analyzing differentially expressed (DE) genes in microarray data. Methods based on statistical tests that incorporate expression level variability are used more commonly than those based on fold change (FC). However, FC based results are more reproducible and biologically relevant.
We propose a new method based on fold change rank ordering statistics (FCROS). We exploit the variation in calculated FC levels using combinatorial pairs of biological conditions in the datasets. A statistic is associated with the ranks of the FC values for each gene, and the resulting probability is used to identify the DE genes within an error level. The FCROS method is deterministic, requires a low computational runtime and also solves the problem of multiple tests which usually arises with microarray datasets.
We compared the performance of FCROS with those of other methods using synthetic and real microarray datasets. We found that FCROS is well suited for DE gene identification from noisy datasets when compared with existing FC based methods.
Differentially expressed genes; Fold change; Averages of ranks; Microarray
Development of the nervous system is finely regulated by consecutive expression of cell-specific transcription factors. Here we show that Helios, a member of the Ikaros transcription factor family, is expressed in ectodermal and neuroectodermal-derived tissues. During embryonic development, Helios is expressed by several brain structures including the lateral ganglionic eminence (LGE, the striatal anlage); the cingulated, insular and retrosplenial cortex; the hippocampus; and the accessory olfactory bulb. Moreover, Helios is also expressed by Purkinje neurons during postnatal cerebellar development. Within the LGE, Helios expression follows a dynamic spatio-temporal pattern starting at embryonic stages (E14.5), peaking at E18.5, and completely disappearing during postnatal development. Helios is expressed by a small population of nestin-positive neural progenitor cells located in the subventricular zone as well as by a larger population of immature neurons distributed throughout the mantle zone. In the later, Helios is preferentially expressed in the matrix compartment, where it colocalizes with Bcl11b and Foxp1, well-known markers of striatal projection neurons. In addition, we observed that Helios expression is not detected in Dlx1/2 and Gsx2 null mutants, while its expression is maintained in Ascl1 mutants. These findings allow us to introduce a new transcription factor in the cascade of events that take part of striatal development postulating the existence of at least 4 different neural progenitors in the LGE. An Ascl1-independent but Gsx2- & Dlx1/2-dependent precursor will express Helios defining a new lineage for a subset of matrix striatal neurons.
The transcription factor FoxP3 partakes dominantly in the specification and function of FoxP3+CD4+ T regulatory cells (Tregs), but is neither strictly necessary nor sufficient to determine the characteristic Treg signature. Computational network inference and experimental testing assessed the contribution of other transcription factors (TF). Enforced expression of Helios or Xbp1 elicited specific signatures, but Eos, Irf4, Satb1, Lef1 and Gata1 elicited exactly the same outcome, synergizing with FoxP3 to activate most of the Treg signature, including key TFs, and enhancing FoxP3 occupancy at its genomic targets. Conversely, the Treg signature was robust to inactivation of any single cofactor. A redundant genetic switch thus locks-in the Treg phenotype, a model which accounts for several aspects of Treg physiology, differentiation and stability.
The Ikaros transcription factor is crucial for many aspects of hematopoiesis. Loss of function mutations in IKZF1, the gene encoding Ikaros, have been implicated in adult and pediatric B cell acute lymphoblastic leukemia (B-ALL). These mutations result in haploinsufficiency of the Ikaros gene in approximately half of the cases. The remaining cases contain more severe or compound mutations that lead to the generation of dominant-negative proteins or complete loss of function. All IKZF1 mutations are associated with a poor prognosis. Here we review the current genetic, clinical and mechanistic evidence for the role of Ikaros as a tumor suppressor in B-ALL.
B cell leukemia; Ikaros; tumor suppressor
Bcl11b is a transcription factor that, within the hematopoietic system, is expressed specifically in T cells. Although Bcl11b is required for T-cell differentiation in newborn Bcl11b-null mice, and for positive selection in the adult thymus of mice bearing a T-cell-targeted deletion, the gene network regulated by Bcl11b in T cells is unclear. We report herein that Bcl11b is a bifunctional transcriptional regulator, which is required for the correct expression of approximately 1000 genes in CD4+CD8+CD3lo double-positive (DP) thymocytes. Bcl11b-deficient DP cells displayed a gene expression program associated with mature CD4+CD8− and CD4− CD8+ single-positive (SP) thymocytes, including upregulation of key transcriptional regulators, such as Zbtb7b and Runx3. Bcl11b interacted with regulatory regions of many dysregulated genes, suggesting a direct role in the transcriptional regulation of these genes. However, inappropriate expression of lineage-associated genes did not result in enhanced differentiation, as deletion of Bcl11b in DP cells prevented development of SP thymocytes, and that of canonical NKT cells. These data establish Bcl11b as a crucial transcriptional regulator in thymocytes, in which Bcl11b functions to prevent the premature expression of genes fundamental to the SP and NKT cell differentiation programs.
Mouse; T-cell differentiation; Transcription factor
Ikaros is a zinc finger transcriptional regulator encoded by the Ikzf1 gene. Ikaros displays crucial functions in the hematopoietic system and its loss of function has been linked to the development of lymphoid leukemia. In particular, Ikaros has been found in recent years to be a major tumor suppressor involved in human B-cell acute lymphoblastic leukemia. Its role in T-cell leukemia, however, has been more controversial. While Ikaros deficiency appears to be very frequent in murine T-cell leukemias, loss of Ikaros appears to be rare in human T-cell acute lymphoblastic leukemia (T-ALL). We review here the evidence linking Ikaros to T-ALL in mouse and human systems.
Ikaros; Notch; T-cell leukemia
The zinc finger transcription factor, Ikaros, is a central regulator of hematopoiesis. It is required for the development of the earliest B cell progenitors and at later stages for VDJ recombination and B cell receptor expression. Mature B cells rely on Ikaros to set the activation threshold for various stimuli, and to choose the correct antibody isotype during class switch recombination. Thus, Ikaros contributes to nearly every level of B cell differentiation and function.
Ikaros; B cells development; B cell activation; Class switch recombination
Although in vitro IL-4 directs CD4 T cells to produce T helper 2 (Th2)-cytokines, these cytokines can be induced in vivo in the absence of IL-4-signalling. Thus, mechanism(s), different from the in vitro pathway for Th2-induction, contribute to in vivo Th2-differentiation. The pathway for in vivo IL-4-independent Th2-differentiation has yet to be characterized.
Helios (ikzf2), a member of the Ikaros transcription regulator family, is expressed in thymocytes and some antigen-matured T cells as well as in regulatory T cells. It has been proposed that Helios is a specific marker for thymus-derived regulatory T cells. Here, we show that mouse ovalbumin-specific CD4 (OTII) cells responding to alum-precipitated ovalbumin (alumOVA) upregulate Th2 features - GATA-3 and IL-4 - as well as Helios mRNA and protein. Helios is also upregulated in follicular helper T (TFh) cells in this response. By contrast, OTII cells responding to the Th1 antigen - live attenuated ovalbumin-expressing Salmonella - upregulate Th1 features - T-bet and IFN-γ - but not Helios. In addition, CD4 T cells induced to produce Th2 cytokines in vitro do not express Helios. The kinetics of Helios mRNA and protein induction mirrors that of GATA-3. The induction of IL-4, IL-13 and CXCR5 by alumOVA requires NF-κB1 and this is also needed for Helios upregulation. Importantly, Helios is induced in Th2 and TFh cells without parallel upregulation of Foxp3. These findings suggested a key role for Helios in Th2 and TFh development in response to alum-protein vaccines. We tested this possibility using Helios-deficient OTII cells and found this deficiency had no discernable impact on Th2 and TFh differentiation in response to alumOVA.
Helios is selectively upregulated in CD4 T cells during Th2 and TFh responses to alum-protein vaccines in vivo, but the functional significance of this upregulation remains uncertain.
Class switch recombination (CSR) allows the humoral immune response to exploit different effector pathways through specific secondary antibody isotypes. However, the molecular mechanisms and factors that control immunoglobulin (Ig) isotype choice for CSR are unclear. We report that deficiency for the Ikaros transcription factor results in increased and ectopic CSR to IgG2b and IgG2a, and reduced CSR to all other isotypes, regardless of stimulation. Ikaros suppresses active chromatin marks, transcription, and activation-induced cytidine deaminase (AID) accessibility at the γ2b and γ2a genes to inhibit class switching to these isotypes. Further, Ikaros directly regulates isotype gene transcription as it directly binds the Igh 3′ enhancer and interacts with isotype gene promoters. Finally, Ikaros-mediated repression of γ2b and γ2a transcription promotes switching to other isotype genes by allowing them to compete for AID-mediated recombination at the single-cell level. Thus, our results reveal transcriptional competition between constant region genes in individual cells to be a critical and general mechanism for isotype specification during CSR. We show that Ikaros is a master regulator of this competition.
Notch activity is essential for early T-cell differentiation, but aberrant activity induces T-cell transformation. Thus, Notch target genes must be efficiently silenced in cells where Notch activity is no longer required. How these genes are repressed remains poorly understood. We report here that the Ikaros transcription factor plays a crucial role in repressing the transcriptional response to Notch signaling in T-cell development. Using the Notch target gene Hes-1 as a model, we show that Ikaros and RBP-Jκ, the transcriptional mediator of Notch signaling, compete for binding to two elements in the Hes-1 promoter in immature thymocytes. This antagonistic interaction likely occurs at the CD4− CD8− CD3− double-negative 4 (DN4) stage, where Ikaros levels and binding to the Hes-1 promoter increase sharply and wild-type thymocytes lose their capacity to transcribe Hes-1 upon Notch stimulation. Nonresponsiveness to Notch signaling requires Ikaros, as Ikaros-deficient DN4 and CD4+ CD8+ double-positive (DP) cells remain competent to express Hes-1 after Notch activation. Further, Hes-1 promoter sequences from Ikaros-deficient DP cells show reduced trimethylated H3K27, a modification associated with silent chromatin. These results indicate that Ikaros functions as a transcriptional checkpoint to repress Notch target gene expression in T cells.
Genome-wide expression profiling of mouse and human leukocytes reveal conserved transcriptional programs of plasmacytoid or conventional dendritic cell subsets.
Dendritic cells (DCs) are a complex group of cells that play a critical role in vertebrate immunity. Lymph-node resident DCs (LN-DCs) are subdivided into conventional DC (cDC) subsets (CD11b and CD8α in mouse; BDCA1 and BDCA3 in human) and plasmacytoid DCs (pDCs). It is currently unclear if these various DC populations belong to a unique hematopoietic lineage and if the subsets identified in the mouse and human systems are evolutionary homologs. To gain novel insights into these questions, we sought conserved genetic signatures for LN-DCs and in vitro derived granulocyte-macrophage colony stimulating factor (GM-CSF) DCs through the analysis of a compendium of genome-wide expression profiles of mouse or human leukocytes.
We show through clustering analysis that all LN-DC subsets form a distinct branch within the leukocyte family tree, and reveal a transcriptomal signature evolutionarily conserved in all LN-DC subsets. Moreover, we identify a large gene expression program shared between mouse and human pDCs, and smaller conserved profiles shared between mouse and human LN-cDC subsets. Importantly, most of these genes have not been previously associated with DC function and many have unknown functions. Finally, we use compendium analysis to re-evaluate the classification of interferon-producing killer DCs, lin-CD16+HLA-DR+ cells and in vitro derived GM-CSF DCs, and show that these cells are more closely linked to natural killer and myeloid cells, respectively.
Our study provides a unique database resource for future investigation of the evolutionarily conserved molecular pathways governing the ontogeny and functions of leukocyte subsets, especially DCs.
Two collections of oligonucleotides have been designed for preparing pangenomic human and mouse microarrays. A total of 148 993 and 121 703 oligonucleotides were designed against human and mouse transcripts. Quality scores were created in order to select 25 342 human and 24 109 mouse oligonucleotides. They correspond to: (i) a BLAST-specificity score; (ii) the number of expressed sequence tags matching each probe; (iii) the distance to the 3′ end of the target mRNA. Scores were also used to compare in silico the two microarrays with commercial microarrays. The sets described here, called RNG/MRC collections, appear at least as specific and sensitive as those from the commercial platforms. The RNG/MRC collections have now been used by an Anglo-French consortium to distribute more than 3500 microarrays to the academic community. Ad hoc identification of tissue-specific transcripts and a ∼80% correlation with hybridizations performed on Affymetrix GeneChip™ suggest that the RNG/MRC microarrays perform well. This work provides a comprehensive open resource for investigators working on human and mouse transcriptomes, as well as a generic method to generate new microarray collections in other organisms. All information related to these probes, as well as additional information about commercial microarrays have been stored in a freely-accessible database called MEDIANTE.
The Ikaros transcription factor is both a key regulator of lymphocyte differentiation and a tumor suppressor in T lymphocytes. Mice carrying a hypomorphic mutation (IkL/L) in the Ikaros gene all develop thymic lymphomas. IkL/L tumors always exhibit strong activation of the Notch pathway, which is required for tumor cell proliferation in vitro. Notch activation occurs early in tumorigenesis and may precede transformation, as ectopic expression of the Notch targets Hes-1 and Deltex-1 is detected in thymocytes from young IkL/L mice with no overt signs of transformation. Notch activation is further amplified by secondary mutations that lead to C-terminal truncations of Notch 1. Strikingly, restoration of Ikaros activity in tumor cells leads to a rapid and specific downregulation of Notch target gene expression and proliferation arrest. Furthermore, Ikaros binds to the Notch-responsive element in the Hes-1 promoter and represses Notch-dependent transcription from this promoter. Thus, Ikaros-mediated repression of Notch target gene expression may play a critical role in defining the tumor suppressor function of this factor.
Retinoid X receptor α–null (RXRα-null) mutants exhibit hypoplasia of their ventricular myocardium and die at the fetal stage. In the present study, we wished to determine whether transgenic re-expression of RXRα in mutant cardiac myocytes could rescue these defects. Two transgenic mouse lines specifically overexpressing an RXRα protein in cardiomyocytes were generated, using the cardiac α-myosin heavy chain (α-MHC) promoter. Breeding the high copy number transgenic line onto an RXRα-null genetic background did not prevent the myocardial hypoplasia and fetal lethality associated with the RXRα–/– genotype, even though the transgene was expressed in the ventricles as early as 10.5 days post-coitum. These data suggest that the RXRα function involved in myocardial growth may correspond to a non–cell-autonomous requirement forsignal orchestrating the growth and differentiation of myocytes. Interestingly, the adult transgenic mice developed a dilated cardiomyopathy, associated with myofibrillar abnormalities and specific deficiencies in respiratory chain complexes I and II, thus providing an additional model for this genetically complex disease.