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J. Biol. Chem., Vol. 282, Issue 9, 6843-6853, March 2, 2007

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Elucidation, Quantitative Refinement, and in Vivo Utilization of the HOXA13 DNA Binding Site^*

Wendy M. Knosp, Chie Saneyoshi, Siming Shou, Hans Peter Bächinger^¶||, and H. Scott Stadler^||1

From the Departments of Molecular and Medical Genetics and ^¶Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, the Department of Anatomy, University of California San Francisco, San Francisco, California 94158-2324, and the ^||Research Division, Shriners Hospital for Children, Portland, Oregon 97239

Mutations in Hoxa13 cause malformations of the appendicular skeleton and genitourinary tract, including digit loss, syndactyly, and hypospadias. To determine the molecular basis for these defects, the DNA sequences bound by HOXA13 were empirically determined, revealing a novel high affinity binding site. Correlating the utilization of this high affinity binding site with genes exhibiting perturbed expression in Hoxa13 mutant limbs, we identified that HOXA13 suppresses the expression of the BMP antagonist, Sostdc1. In the absence of HOXA13 function, Sostdc1 is ectopically expressed in the distal limb, causing reduced expression of BMP-activated genes and decreased SMAD phosphorylation. Limb chromatin immunoprecipitation revealed HOXA13 binding at its high affinity site in two conserved Sostdc1 regulatory sites in vivo. In vitro, HOXA13 represses gene expression through the Sostdc1 high affinity binding sites in a dosage-dependent manner. Together, these findings confirm that the high affinity HOXA13 binding site deduced by quantitative analyses is used in vivo to facilitate HOXA13 target gene regulation, providing a critical advance toward understanding the molecular basis for defects associated with the loss of HOXA13 function.

Received for publication, November 21, 2006

^* This work was supported by grants from the Shriners Hospital for Children (to H. S. S. and H. P. B.), National Institutes of Health Grants 5R01DK066539-01 (to H. S. S.) 5T32GM008617-09 (to W. M. K.), and an N. L. Tartar Research Fellowship (to W. M. K.). Biostatical and Affymetrix microarray support was provided in part by the Biostatistics Shared Resource of the Oregon Health and Science University Cancer Institute (Grant P30 CA69533). 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. 1 and Tables 1 and 2.

¹ To whom correspondence should be addressed: Shriners Hospital for Children, 3101 S.W. Sam Jackson Park Rd., Portland, OR 97239. Tel.: 503-221-3447; E-mail: hss{at}shcc.org.