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.