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J. Biol. Chem., Vol. 278, Issue 30, 27532-27539, July 25, 2003

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Characterization of Nkx3.2 DNA Binding Specificity and Its Requirement for Somitic Chondrogenesis^*

Dae-Won Kim , Hervé Kempf , Raymond E. Chen and Andrew B. Lassar ¶

From the Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

We have previously shown that Nkx3.2, a member of the NK class of homeoproteins, functions as a transcriptional repressor to promote somitic chondrogenesis. However, it has not been addressed whether Nkx3.2 can bind to DNA in a sequence-specific manner and whether DNA binding by Nkx3.2 is required for its biological activity. In this work, we employed a DNA binding site selection assay, which identified TAAGTG as a high affinity Nkx3.2 binding sequence. Sequence-specific binding of Nkx3.2 to the TAAGTG motif in vitro was confirmed by electrophoretic mobility shift assays, and mutagenesis of this sequence revealed that HRAGTG (where H represents A, C, or T, and R represents A or G) comprises the consensus DNA binding site for Nkx3.2. Consistent with these findings, the expression of a reporter gene containing reiterated Nkx3.2 binding sites was repressed in vivo by Nkx3.2 co-expression. In addition, we have generated a DNA nonbinding point mutant of Nkx3.2 (Nkx3.2-N200Q), which contains an asparagine to glutamine missense mutation in the homeodomain. Interestingly, despite being defective in DNA binding, Nkx3.2-N200Q still retains its intrinsic transcriptional repressor function. Finally, we demonstrate that unlike wild-type Nkx3.2, Nkx3.2-N200Q is unable to activate the chondrocyte differentiation program in somitic mesoderm, indicating that DNA binding by Nkx3.2 is critical for this factor to induce somitic chondrogenesis.

Received for publication, February 11, 2003 , and in revised form, April 14, 2003.

^* This work was funded by grants from the National Institutes of Health (to A. B. L.). 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.

Supported by a fellowship from the Arthritis Foundation.

Supported by a fellowship from ARC and a long-term fellowship from the Human Frontier Science Program and a recipient of a Fondation Bettencourt Schueller award.

^¶ To whom correspondence should be addressed: Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-3831; Fax: 617-738-0516; E-mail: andrew_lassar{at}hms.harvard.edu.

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