The Ikaros protein

1.3.1.1 Discovery

Hematopoiesis is the process of producing a functional distinct set of cells that comprise the mature blood. These cells arise from pluripotent hematopoietic stem cells that successively become more specified by regulated division and differentiation steps. This process is tightly

the transition of hematopoietic precursors to a highly differentiated cell (reviewed in Orkin, 1995; and in Cantor and Orkin, 2001). The regulation of this coordinated program of gene activation and silencing is mediated by transcription factors and the search for such factors started more then 15 years ago. In an attempt to isolate regulatory proteins involved in the control of differentiation of the hematopoietic T cell lineage, Georgopoulos and co-workers (1992) identified the Ikaros protein. The T cell lineage is characterized by the presence of the CD3-T cell receptor complex which is encoded by the CD3δ gene (Furley et al., 1986;

Haynes et al., 1989). Ikaros was isolated as a factor that specifically binds to a G-rich sequence present in the regulatory elements of CD3δ (Georgopoulos et al., 1992). This factor was also shown to encode the Lymphoid transcription factor 1 (LyF-1) protein, which binds to functionally important regulatory sites within the lymphocyte specific terminal deoxynucleotidyltransferase (TdT) promoter (Lo et al., 1991; Ernst et al., 1993; Hahm et al., 1994). To further establish its function as a factor involved in the development of the hematopoietic system, the expression pattern of the Ikaros protein was analyzed. Ikaros is first detected in hematopoietic precursor populations and is later mainly present in mature T cells, B-cells and natural killer cells. In contrast, it is downregulated in most differentiated erythroid and myeloid lineages including mature monocytes, macrophages and erythrocytes (Georgopoulos et al., 1992; Klug et al., 1998). Thus, the temporal expression pattern of Ikaros in hematopoietic cell lines is consistent with the idea that it plays a role in lymphoid cell development Georgopoulos et al., 1994; Klug et al., 1998; reviewed by Georgopoulos et al., 1997; Westman et al., 2002; and by Cobb and Smale, 2005).

1.3.1.2 Ikaros isoforms

Ikaros is composed of seven exons from which at least eight isoforms (Ik-1 to Ik-8, Figure 1.4) can be generated by alternative mRNA splicing events (Hahm et al., 1994; Molnar and Georgopoulos, 1994; Molnar et al., 1996). As shown in Figure 1.4, each isoform encodes a distinct C2H2 ZF protein and most of these isoforms consist of two defined C2H2 ZF domains. While the C-terminal C2H2 ZF domain is present in all isoforms, the N-terminal C2H2 ZF domain contains different combinations of one to four C2H2 ZFs and two isoforms (Ik-6 and Ik-8) have no terminal C2H2 ZFs at all. It has been demonstrated that the N-terminal C2H2 ZFs are required for DNA binding. In addition, DNA specificity of the different isoforms was analyzed extensively using gel-shift assays and PCR site selections (Molnar and Georgopoulos, 1994) which showed that three N-terminal C2H2 ZFs are

necessary to bind to a single binding site of the conserved core motif GGGAA. Thus, only three of the Ikaros proteins (Ik-1, Ik-2 and Ik-3) which contain three or four N-terminal C2H2 ZFs, bind to this sequence. Ik-4 is composed of two N-terminal C2H2 ZFs and binds to a tandem recognition site containing an inverted repeat of the consensus motif. On the other hand, Ikaros isoforms with only one or no N-terminal C2H2 ZFs (Ik-5, Ik-6, Ik-7 and Ik-8) are not able to mediate interactions with DNA (Molnar and Georgopoulos, 1994).

The C-terminal C2H2 ZF domain present in all Ikaros isoforms has been shown to mediate dimerization (Sun et al., 1996; McCarty et al., 2003) and is utilized to engage these various Ikaros proteins in homo- and heterodimeric complexes. Oligomerization between isoforms composed of N-terminal C2H2 ZFs capable of DNA-binding dramatically increases their DNA affinity. However, heterodimers consisting of Ikaros proteins with and without a functional DNA-binding domain can not bind to DNA. Furthermore, the formation of such complexes interferes with the transcriptional activity of DNA-binding isoforms which could provide a mechanism to control activity of these isoforms (Sun et al., 1996).

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Figure 1.4 Schematic diagram of the Ikaros isoforms (Ik-1 - Ik-8). Exons are presented as white numbered boxes and individual ZFs are depicted as grey rounded rectangles. ZF domains involved in DNA-binding and dimerization are also indicated.

1.3.1.3 Knock out studies of Ikaros

To evaluate the role of Ikaros in lymphoid cell development, knock out studies in mice were performed. Mice homozygous for a deletion of the N-terminal DNA-binding domain completely lack lymphoid progenitors as well as mature B and T lymphocytes and natural killer cells (Georgopoulos et al., 1994). On the other hand, mice that are heterozygous for this mutation generate abnormal T cells and develop T-cell leukemias and lymphomas (Winandy et al., 1995). This suggests that Ikaros is necessary for early development of lymphoid progenitors but also plays a role in later T cell maturation. Interestingly, mice homozygous for a deletion of the C-terminal C2H2 ZF domain in Ikaros display a phenotype less severe than the phenotype caused by the missing N-terminal domain (Wang et al., 1996). This deletion is considered to be a null mutation resulting in a complete loss of Ikaros activity while the deletion of the N-terminal DNA binding domain is believed to fulfill a dominant-negative function. Thus, the severe phenotype observed in mice homozygous for the N-terminal C2H2 ZF domain can be explained by a mechanism in which this mutated protein dominantly effects and disrupts other proteins that could otherwise partially compensate for a loss of function of the Ikaros protein (see next section).