Targeting of Ikaros to pericentromeric heterochromatin by direct DNA binding

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Abstract
Ikaros is a sequence-specific DNA-binding protein that is essential for lymphocyte development. Little is known about the molecular function of Ikaros, although recent results have led to the hypothesis that it recruits genes destined for heritable inactivation to foci containing pericentromeric heterochromatin. To gain further insight into the functions of Ikaros, we have examined the mechanism by which it is targeted to centromeric foci. Efficient targeting of Ikaros was observed upon ectopic expression in 3T3 fibroblasts, demonstrating that lymphocyte-specific proteins and a lymphoid nuclear architecture are not required. Pericentromeric targeting did not result from an interaction with the Mi-2 remodeling factor, as only a small percentage of Mi-2 localized to centromeric foci in 3T3 cells. Rather, targeting was dependent on the amino-terminal DNA-binding zinc finger domain and carboxy-terminal dimerization domain of Ikaros. The carboxy-terminal domain was required only for homodimerization, as targeting was restored when this domain was replaced with a leucine zipper. Surprisingly, a detailed substitution mutant analysis of the amino-terminal domain revealed a close correlation between DNA-binding and pericentromeric targeting. These results show that DNA binding is essential for the pericentromeric localization of Ikaros, perhaps consistent with the presence of Ikaros binding sites within centromeric DNA repeats. Models for the function of Ikaros that are consistent with this targeting mechanism are discussed.