Zinc Finger Proteins: From Atomic Contact to Cellular Function, edited by Shiro Iuchi and Natalie Kuldell. ©2004 Landes Bioscience/Eurekah.com. CHAPTER 27 The Role of the Ikaros Gene Family in Lymphocyte Development Pablo Gomez-del Arco, Taku Naito, John Seavitt, Toshimi Yoshida, Christine Williams and Katia Georgopoulos Abstract I n many developmental systems, nuclear regulators have been implicated in coupling key events in gene expression with specific cell fate and lineage decisions. In the hemo-lymphoid system, the Ikaros gene family of zinc finger DNA binding fac- tors control lymphocyte specification and homeostasis from the hemopoietic stem cell (HSC) throughout development. The de- pendence of hemo-lymphoid differentiation on Ikaros DNA binding activity together with the presence of Ikaros proteins within higher order chromatin remodeling complexes supports the hypothesis that Ikaros plays a key role in the lineage-specific remodeling of chromatin. Association of Ikaros and its remodel- ing partners with the chromatin of key lineage-specific genes, and the dependence of these genes on Ikaros complexes for their expression supports this hypothesis and provides unique para- digms to study chromatin regulation of differentiation. The Ikaros-gene Family Functional Domains of Ikaros Gene Family The Ikaros gene family encodes a unique group of DNA-binding proteins characterized by the presence of two func- tionally distinct domains containing Krüppel-type zinc fingers. 1-3 Initially identified as a regulator of lymphoid-specific genes, Ikaros was subsequently determined to be the archetype for a family of proteins including Aiolos, Helios, and Eos, expression of which extends from hemo-lymphoid to neuro-epithelial tissues (Fig. 1). 4-7 cDNAs for the Ikaros family members have been isolated from human, mouse, chicken, amphibian, fish, and protochordate species and comparison of the protein sequences indicates a strong conservation of domain function. 8,9 The full length Ikaros protein is characterized by an amino-terminal zinc finger-dependent DNA binding domain and a carboxy-terminal zinc finger domain required for multimer as- sembly (Fig. 1). 2,3 Ikaros is comprised of two alternatively used noncoding exons followed by seven coding exons. 10 Alternative splicing of exons four through seven gives rise to ten recognized isoforms. These isoforms vary in the inclusion of the four amino-terminal zinc fingers required for DNA binding. Mutagen- esis studies indicate that the function and presence of the core pair of zinc fingers, F2 and F3, are critical for high affinity bind- ing to the core motif 5'-c/TGGGAAT/c-3'. 2,11 The presence of at least one additional flanking zinc finger, F1 or F4, further in- fluences binding to this motif. 2 The amino-terminal zinc finger domain is also required for nuclear localization. 2,11 All recognized splicing isoforms retain the carboxy-terminal pair of zinc fingers. This region may represent an evolutionarily conserved interac- tion module, as it shares homology with the Drosophila hunch- back and mammalian Pegasus and tricho-rhino-phalangeal TRPS1 proteins. 1,12-14 This multimerization domain allows assembly between Ikaros proteins and other family members. Multimerization between Ikaros isoforms that can and cannot bind DNA compromises the ability of the resulting complex to bind DNA. This suggests that the nonbinding isoforms may rep- resent a naturally occurring dominant negative mechanism to regulate the activity of this family of proteins. 15 Nevertheless, the DNA-binding isoforms Ik1 and Ik2 are most abundant in nor- mal lymphocytes, although increased expression of the nonDNA Figure 1. Ikaros family proteins. Schematic representation of functional domains in Ikaros family mem- ber proteins and various splicing isoforms. Exons are illustrated indi- vidually with zinc fingers shown as boxes. Regions required for DNA-binding and multimerization are indicated.