Ikaros fingers on lymphocyte differentiation

Abstract

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 (NuRD) complex 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.

Figure 1. Schematic representation of the Ikaros isoforms, DNA binding motifs and of the Ikaros-NuRD complex and its mode of action in lymphocytes

(a) Exon composition containing Zn finger motifs involved in DNA binding and protein dimerization is shown for Ikaros isoforms and Ikaros family members (Aiolos, Helios and Eos). Exons are shown as light blue boxes. Dark blue bars indicate zinc fingers. (b) Transcription factor binding motifs identified in the vicinity of Ikaros enrichment peaks at enhancer regions in thymocytes. Two highly enriched Ikaros binding motifs identified by de novo motif search on its chromatin binding sites. (c) Structure of the Ikaros-NuRD complex and of Mi-2β. The NuRD complex contains Class I histone deacetylases (HDAC1/2) and the ATP-dependent chromatin remodeler Mi-2β (and α). (d) A model of negative and positive regulation by the NuRD complex. Targeting of the Mi-2β–NuRD complex to permissive chromatin (H3K4me3, H3K9Ac) is restricted to lymphoid genes by the Ikaros family proteins. Our hypothesis is that NuRD’s repressive activities are poised by Ikaros extensive DNA binding at its target sites. Upon reduction in Ikaros activity either through posttranslational modification of the protein or through Ikaros inactivating mutations increases chromatin access of the Mi-2β–NuRD complex and loss of lymphoid gene expression. Upon loss of Ikaros, the NuRD complex also re-distributes to new sites associated with promoters of transcriptionally poised genes that support cell growth, proliferation and metabolism causing their activation in part by displacing the PRC2 complex. Ik, Ikaros; Aio, Aiolos; polII, RNA polymerase II; PRC, polycomb repressive complex.

Figure 2. Cellular and transcriptional hierarchies in early hematopoiesis

(a) The lineage potential of each progenitor in the hematopoietic hierarchy is denoted by letter size. B, B cells; T, T cells; E, erythroid; M, myeloid. Relative changes in Ikaros expression in this developmental hierarchy is shown at the bottom. Red arrows indicate critical stages where Ikaros family proteins are up-regulated. (b) The two cascades of lineage specific gene programs that originate in the multi-potent HSC and segregate within the erythroid and lympho-myeloid, pathways are shown. The early lympho-myeloid cascade is comprised of two layers of gene expression, the s-myly is primed in the HSC and further induced in the LMPP and downstream progeny whereas r-myly is primed in the LMPP. In sharp contrast, only one layer is seen in the early erythroid cascade reflecting a lack of progressive lineage restriction points. (c) Effects of Ikaros deficiency on the stem and s-myly signatures. A heatmap of gene expression of the s-myly and stem cell signatures in the HSC, LMPP, GMP, MEP and proB from WT and Ikaros null mice is shown. The signature designation is provided on the right side. The red bars indicate the up-regulated genes and the green bar indicates the down-regulated genes upon loss of Ikaros. The leg of the s-myly signature that is down-regulated is highly enriched for lymphoid promoting genes, whereas the middle section of the s-myly signature that is up-regulated is highly enriched for myeloid promoting growth factors and transcription regulators. Examples of the deregulated genes are provided in each box.

Figure 3. Effects of Ikaros mutations in early lymphoid development and homeostasis

(a) A summary of Ikaros’ roles in early lymphocyte differentiation as revealed by various mouse genetic models. ΔIk, germline Ikaros null mutation; Ikdn. germline Ikaros dominant negative mutation; IkE5^Δ/Δ, B cell specific conditional Ikaros dominant negative mutation. (b) A diagram of interactions between integrin, IL-7R and pre-BCR signaling in WT large pre-B cells and the effects of an Ikaros dominant negative mutation (IkE5^Δ/Δ) on this signaling network. The strength of the effect of individual signaling pathways on cellular properties such as survival, self-renewal and proliferation is depicted by letter size. Green arrows and red bars indicate positive and negative interactions respectively. pFAK, phosphorylated FAK; IL-7R, Interleukin-7 receptor; GHR, growth factor and/or cytokine receptor.

Figure 4. A summary of regulatory elements, their activities and stage specificity at the Ikaros gene (Ikzf1)

(a) A summary of the activities of the regions tested in transgenic mouse models (C, D, E, F, G, H, I, J) at the Ikzf1 locus [34,62,63]. Two promoter regions (A and B) and a promoter element (p) are shown. (b) A diagram of the activities of the lympho-myeloid promoter B and enhancers during lymphopoiesis. Promoter B (+p) provides Ikaros expression in the HSC, B cells, myeloid and DN1-3 thymocytes, however, enhancers J, E, F, H, I are required to maintain the expression in restricted chromatin environments. The enhancer C(D) is required for up-regulation of Ikaros from the HSC to the LMPP and it can further maintain the expression throughout T cell development. Although enhancer H cannot provide activity in the LMPP, it can support the expression past DN3 stages.