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J. Biol. Chem., Vol. 283, Issue 13, 8291-8300, March 28, 2008

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Recruitment of Ikaros to Pericentromeric Heterochromatin Is Regulated by Phosphorylation^*

Zafer Gurel, Tapani Ronni, Sam Ho, Jason Kuchar¹, Kimberly J. Payne^¶, Christoph W. Turk^||, and Sinisa Dovat²

From the Department of Pediatrics, University of Wisconsin, Madison, Wisconsin 53792-4108, Michigan State University, East Lansing, Michigan 48824, the ^¶Center for Health Disparities and Molecular Medicine and the Department of Pathology and Human Anatomy, Loma Linda University School of Medicine, Loma Linda, California 92350, and ^||Proteomics and Biomarkers, Max Planck Institute of Psychiatry, Munich D-80804, Germany

Ikaros encodes a zinc finger protein that is involved in heritable gene silencing. In hematopoietic cells, Ikaros localizes to pericentromeric heterochromatin (PC-HC) where it recruits its target genes, resulting in their activation or repression via chromatin remodeling. The function of Ikaros is controlled by post-translational modifications. CK2 kinase has been shown to phosphorylate Ikaros at its C terminus, affecting cell cycle progression. Using in vivo labeling of murine thymocytes followed by phosphopeptide mapping, we identified four novel Ikaros phosphorylation sites. Functional analysis of phosphomimetic mutants showed that the phosphorylation of individual amino acids determines the affinity of Ikaros toward probes derived from PC-HC. In vivo experiments demonstrated that targeting of Ikaros to PC-HC is regulated by phosphorylation. The ability of Ikaros to bind the upstream regulatory elements of its known target gene terminal deoxynucleotidyltransferase (TdT) was decreased by phosphorylation of two amino acids. In thymocytes, Ikaros acts as a repressor of the TdT gene. Induction of differentiation of thymocytes with phorbol 12-myristate 13-acetate plus ionomycin results in transcriptional repression of TdT expression. This process has been associated with increased binding of Ikaros to the upstream regulatory element of TdT. Phosphopeptide analysis of in vivo-labeled thymocytes revealed that Ikaros undergoes dephosphorylation during induction of thymocyte differentiation and that dephosphorylation is responsible for increased DNA binding affinity of Ikaros toward the TdT promoter. We propose a model whereby reversible phosphorylation of Ikaros at specific amino acids controls the subcellular localization of Ikaros as well as its ability to regulate TdT expression during thymocyte differentiation.

Received for publication, September 20, 2007 , and in revised form, January 3, 2008.

^* This work was supported by National Institutes of Health Grants K22 CA 111392 (to S. D.) and 5K01 DK066163 (to K. J. P.), a Midwest Athletes Against Childhood Cancer grant award, and a Cure Kids Cancer Coalition grant (to S. D.). This work was also supported by the University of Wisconsin Medical Education and Research Committee New Investigator Program (to S. D.). 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.

¹ Current address: Thermo Fisher Scientific, Schaumburg, IL 60010.

² To whom correspondence should be addressed: University of Wisconsin, Dept. of Pediatrics, Division of Pediatric Hematology/Oncology, 600 Highland Ave., H4/431 CSC Madison, WI 53792-4108. Tel.: 608-262-2415; E-mail: dovat{at}pediatrics.wisc.edu.

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