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J. Biol. Chem., Vol. 281, Issue 47, 35747-35756, November 24, 2006

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Characterization of NOBOX DNA Binding Specificity and Its Regulation of Gdf9 and Pou5f1 Promoters^*

From the Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas 77030

Nobox (newborn ovary homeobox gene) deficiency disrupts early folliculogenesis and the expression of oocyte-specific genes in mice. Here, we identified several cis-acting sites, TAATTG, TAGTTG, and TAATTA as NOBOX DNA binding elements (NBEs) using a library of randomly generated oligonucleotides by cyclic amplification of sequence target assay and mutation analyses. We show that NOBOX preferentially binds to the NOBOX binding elements with high affinity. In addition, we found that promoter regions of mouse Pou5f1 and Gdf9 contain one (–426) and three NOBOX binding elements (–786, –967, and –1259), respectively. NOBOX binds to these putative NOBOX binding elements with high affinity and augmented transcriptional activity of luciferase reporter driven by mouse Pou5f1 and Gdf9 promoters containing the NOBOX binding elements. In chromatin immunoprecipitation assays, DNA sequences from Pou5f1 and Gdf9 promoters co-precipitated with anti-NOBOX antibody. These results suggest that NOBOX directly regulates the transcription of Pou5f1 and Gdf9 in oocytes during early folliculogenesis.

Received for publication, April 26, 2006 , and in revised form, September 13, 2006.

^* This work was supported by Grant HD44858 from the National Institutes of Health and a March of Dimes Basil O'Connor Award (5-FY02–266) (to A. R.). 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 To whom correspondence should be addressed: Dept. of Obstetrics and Gynecology, Baylor College of Medicine 1709 Dryden St., Suite 1100, Houston, TX 77030. Tel.: 713-798-1049; Fax: 713-798-2744; E-mail: rajkovic{at}bcm.edu.

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