Ikaros is expressed in early hematopoietic progenitors and is required for lymphoid differentiation. Analysis of Ikaros null populations revealed a lack of defining markers for early fate-restricted progenitors, but it was difficult to discern whether Ikaros was required for formation of these populations, or for expression of these markers. Here we use a GFP reporter based on Ikaros regulatory elements to identify the HSC and separate early progenitors in both wild-type and Ikaros-null mice. The presence of lympho-myeloid progenitors is revealed in Ikaros-null mice, which lack the defining factor Flt3 and are capable of myeloid, but not lymphoid differentiation. In contrast, lack of Ikaros in the common myeloid progenitor results in increased formation of erythro-megakaryocyte at the expense of myeloid progenitors and influences their subsequent differentiation. By this approach, pivotal roles for Ikaros in distinct fate decisions in the early hematopoietic hierarchy are revealed. KeywordsIkaros-reporter; hematopoiesis; progenitors; cell fateThe long-term hematopoietic stem cell (HSC), capable of self-renewal and differentiation into a number of distinct lineages, is responsible for the lifelong generation of all blood and immune cell types 1-3 . Prospective isolation of HSCs and progenitor populations with conventional cell surface markers has identified rare, multipotent cells with defined lineage activities that in turn have been used to infer prevailing models of lineage restriction 4-6 . For example, the isolation of a common myeloid progenitor (CMP) and a common lymphoid progenitor (CLP), considered to be the respective roots of the erytho-myeloid and lymphoid lineages, has lent support to an early and strict separation of the lymphoid from the erythromyeloid pathways.The HSC compartment is operationally defined within the LinSca-1 hi c-Kit hi (LSK) population that constitutes 0.1% of the adult bone marrow (BM) cells and contains both long-term (LT) and short-term (ST) HSC-also known as multipotent progenitors (MPP) 7,8 . Use of additional markers, including CD34 and Flt3, has separated LT-HSCs (Lin − Sca-1 hi cKit hi CD34 − Flt3 neg-lo ) from ST-HSCs (Lin − Sca-1 hi c-Kit hi CD34 + Flt3 neg-lo ) and more short-lived lymphoid-primed progenitors (Lin − Sca-1 hi c-Kit hi CD34 + Flt3 + ) 9-12 .Restricted erythro-myeloid progenitors are present within the more abundant Lin − Sca-1 − cKit hi (LK) population (0.6-1% of the BM) that can be further subdivided into a common myeloid progenitor (CMP, CD34 + FcγR lo ) and its more restricted progeny of megakaryoerythrocyte (MEP, CD34 − FcγR lo ) and granulo-monocyte (GMP, CD34 + FcγR hi ) progenitors. A restricted common lymphoid progenitor (CLP) capable of B, T and natural killer (NK)

Summary Here we investigate the mechanisms that underlie the induction of developmental potential and establishment of cell fate during early hematopoiesis. A cascade of lineage-affiliated gene expression signatures, primed in hematopoietic stem cells (HSC) and differentially propagated in lineage-restricted progenitors, is identified. First evidence is provided for a stochastic sampling of lymphoid, erythroid and myeloid transcripts in HSC and multipotent progenitors (MPP). Multi-lineage priming is subsequently resolved upon lineage restrictions. Nonetheless, an unexpected association of lymphoid and myeloid signatures is detected past a nominal myeloid restriction point and a previously unappreciated lymphoid potential is revealed for this stage in development. New insight is provided into Ikaros' role as a bivalent regulator of multi-lineage priming during early hematopoiesis. Whereas Ikaros is responsible for activation of a cascade of lymphoid signatures in the HSC, at subsequent restriction points it is also involved in the repression of lineage-inappropriate signatures including stem cell-specific genes.

Cell fate decisions depend on the interplay between chromatin regulators and transcription factors. Here we show that activity of the Mi-2β nucleosome remodeling and deacetylase (NuRD) complex was controlled by the Ikaros family of lymphoid-lineage determining proteins. Ikaros, an integral component of the NuRD complex in lymphocytes, tethered this complex to active lymphoid differentiation genes. Loss in Ikaros DNA binding activity caused a local increase in Mi-2β chromatin remodeling and histone deacetylation and suppression of lymphoid gene expression. The NuRD complex also redistributed to transcriptionally poised non-Ikaros gene targets, involved in proliferation and metabolism, inducing their reactivation. Thus, release of NuRD from Ikaros regulation blocks lymphocyte maturation and mediates progression to a leukemic state by engaging functionally opposing epigenetic and genetic networks.

Deletion of the Ikaros (Ikzf1) DNA-binding domain generates dominant-negative isoforms that interfere with Ikaros family activity and correlate with poor prognosis in human precursor B cell acute lymphoblastic leukemias (B-ALL). Here, we show that conditional inactivation of the Ikaros DNA binding domain in early pre-B cells arrests their differentiation at a stage where integrin-dependent niche adhesion augments mitogen-activated protein kinase signaling, proliferation, and self-renewal, and attenuates pre-B cell receptor signaling and differentiation. Transplantation of polyclonal Ikzf1 mutant pre-B cells results in long-latency oligoclonal pre-B-ALL, demonstrating that loss of Ikaros contributes to multistep B-leukemogenesis. These results explain how normal pre-B cells transit from a highly proliferative and stromal-dependent to a stromal-independent phase where differentiation is enabled, providing potential therapeutic strategies for IKZF1 mutant B-ALL.

The ability of somatic stem cells to self-renew and differentiate into downstream lineages is dependent on specialized chromatin environments that keep stem cell-specific genes active and key differentiation factors repressed but poised for activation. The epigenetic factors that provide this type of regulation remain ill-defined. Here we provide the first evidence that the SNF2-like ATPase Mi-2␤ of the Nucleosome Remodeling Deacetylase (NuRD) complex is required for maintenance of and multilineage differentiation in the early hematopoietic hierarchy. Shortly after conditional inactivation of Mi-2␤, there is an increase in cycling and a decrease in quiescence in an HSC (hematopoietic stem cell)-enriched bone marrow population. These cycling mutant cells readily differentiate into the erythroid lineage but not into the myeloid and lymphoid lineages. Together, these effects result in an initial expansion of mutant HSC and erythroid progenitors that are later depleted as more differentiated proerythroblasts accumulate at hematopoietic sites exhibiting features of erythroid leukemia. Examination of gene expression in the mutant HSC reveals changes in the expression of genes associated with self-renewal and lineage priming and a pivotal role of Mi-2␤ in their regulation. Thus, Mi-2␤ provides the hematopoietic system with immune cell capabilities as well as with an extensive regenerative capacity.[Keywords: Mi-2␤; chromatin; HSC; multipotency; self-renewal; lineage priming] Supplemental material is available at http://www.genesdev.org. Received December 13, 2007; revised version accepted March 4, 2008. The defining properties of somatic stem cells, their ability to self-renew and to progress through available differentiation pathways, are critical for the life-long tissue integrity of multicellular organisms (Weissman 2000;Lemischka and Moore 2003). A balance between stem cell quiescence and activation is required to sustain the stem cell pool and to provide adequate numbers of mature cells to meet normal homeostatic conditions. Both the self-renewal and differentiation properties of stem cells can be altered dramatically in order to meet demands imposed by stress conditions.In hematopoietic tissue, the most primitive stem cells are thought to be in a comparatively quiescent state. They cycle with slow kinetics that strongly correlate with their long-term self-renewing potential (LT-HSC) (Morrison and Weissman 1994;Cheshier et al. 1999). Thus, maintenance of hematopoietic stem cell (HSC) activity can be compromised in two ways. On the one hand, a cell cycle block can prevent self-renewing divisions. On the other hand, prolonged cell cycle activation can lead to HSC exhaustion. This has been corroborated by studies on components of the cell cycle machinery and on signaling pathways that modulate their activity. An increase in expression of the cell cycle inhibitors p16 Ink4A, p19Ink4D/Arf , or p18Ink4C has an adverse effect on the HSC's self-renewal, presumably by restricting its entry into the cell cycle (Park et a...

We generated interleukin-5 receptor alpha chain (IL-5R alpha)-deficient (IL-5R alpha-/-) mice by gene targeting. The IL-5R alpha-/- mice showed decreased numbers of B-1 cells concomitant with low serum concentrations of IgM and IgG3. They showed no IL-5-induced enhancement of B cell responses to T-independent antigens. The number of alpha beta T cell receptor-positive thymocytes tended to decrease in 3-week-old IL-5R alpha-/- mice, returning to normal by 6 weeks of age. The IL-5R alpha-/- mice produced basal levels of eosinophils, while their bone marrow cells failed to form eosinophilic colonies in response to IL-5. Impaired eosinophilopoiesis in IL-5R alpha-/-mice enhanced the survival of Angiostrongylus cantonensis. These results indicate that IL-5-induced eosinophils serve as potent effector cells in the killing of Angiostrongylus cantonensis in mice.

SUMMARY Precise control of gene expression plays fundamental roles in brain development, but the roles of chromatin regulators in neuronal connectivity have remained poorly understood. We report that depletion of the NuRD complex by in vivo RNAi and conditional knockout of the core NuRD subunit Chd4 profoundly impairs the establishment of granule neuron parallel fiber/Purkinje cell synapses in the rodent cerebellar cortex in vivo. By interfacing genome-wide sequencing of transcripts and ChIP-Seq analyses, we uncover a network of repressed genes and distinct histone modifications at target gene promoters that are developmentally regulated by the NuRD complex in the cerebellum in vivo. Finally, in a targeted in vivo RNAi screen of NuRD target genes, we identify a program of NuRD-repressed genes that operate as critical regulators of presynaptic differentiation in the cerebellar cortex. Our findings define NuRD-dependent promoter decommissioning as a developmentally-regulated programming mechanism that drives synaptic connectivity in the mammalian brain.

The Ikaros family of DNA binding proteins are critical regulators of lymphocyte differentiation. In multipotent hematopoietic progenitors, Ikaros supports transcriptional priming of genes promoting lymphocyte differentiation. Ikaros targets the Nucleosome Remodeling Deacetylase complex (NuRD) to lymphoid lineage genes, thereby increasing chromatin accessibility and transcriptional priming. After lymphoid lineage specification, Ikaros expression is raised to levels characteristic of intermediate B cell and T cell precursors, which is necessary to support maturation and prevent leukemogenesis. Loss of Ikaros in T cell precursors allows the NuRD complex to repress lymphocyte genes and extends its targeting to genes that support growth and proliferation, causing their activation and triggering a cascade of events that leads to leukemogenesis. Loss of Ikaros in B cell precursors blocks differentiation and perpetuates stromal adhesion by enhancing integrin signaling. The combination of integrin and cytokine signaling in Ikaros-deficient pre-B cells promotes their survival and self-renewal. The stages of lymphocyte differentiation that are highly dependent on Ikaros are underscored by changes in Ikaros transcription, supported by a complex network of stage-specific regulatory networks that converge upon the Ikzf1 locus. It is increasingly apparent that understanding the regulatory networks that operate upstream and downstream of Ikaros is critical not only for our understanding of normal lymphopoiesis, but also in placing the right finger on the mechanisms that support hematopoietic malignancies in mouse and human.