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J. Biol. Chem., Vol. 282, Issue 41, 30227-30238, October 12, 2007

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Predominant Interaction of Both Ikaros and Helios with the NuRD Complex in Immature Thymocytes^*

From the Howard Hughes Medical Institute and Department of Microbiology, Immunology, and Molecular Genetics, UCLA, Los Angeles, California 90095

Ikaros is the founding member of a small family of C2H2 zinc-finger DNA-binding proteins that carry out critical functions during lymphocyte development. Although interactions between Ikaros and various proteins have been reported, Ikaros-containing complexes have not been purified to determine their composition and identify the predominant interacting partners. In this study, a tandem affinity purification-mass spectrometry strategy was developed for the isolation of complexes formed by Ikaros and by Helios, a T-cell-restricted member of the Ikaros family that remains largely uncharacterized. This strategy, which appears to be well suited for general use in mammalian cells, relies on an N-terminal polypeptide containing a double FLAG epitope, followed by a tobacco etch virus protease cleavage site and calmodulin binding peptide. In extracts from a murine thymocyte line, Ikaros and Helios associated under moderate stringency conditions only with other members of the Ikaros family. However, under low stringency conditions, both tagged proteins assembled into higher molecular weight complexes. Mass spectrometry revealed that both proteins associated predominantly with subunits of NuRD, an ATP-dependent nucleosome remodeling complex implicated in transcriptional repression and activation and previously reported to associate with Ikaros. Further analysis of the affinity-purified Ikaros revealed that several serines and threonines are phosphorylated in the thymocyte line, with apparent changes upon thymocyte maturation. These results support the hypothesis that the NuRD complex makes major contributions to the functions of both Ikaros and Helios and that the activities of these proteins may be regulated in part by changes in phosphorylation.

Received for publication, March 23, 2007 , and in revised form, July 9, 2007.

^* This work was supported in part by National Institutes of Health Grant R01 DK43736. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and Table S1.

¹ To whom correspondence should be addressed: Dept. of Microbiology, Immunology, and Molecular Genetics, 6-730 MRL, 675 Charles E. Young Drive South, UCLA, Los Angeles, CA 90095-1662. Tel.: 310-206-4777; Fax: 310-206-8623; E-mail: smale{at}mednet.ucla.edu.

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