Characterization of NOBOX DNA Binding Specificity and Its Regulation of Gdf9 and Pou5f1 Promoters*
- 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.
Abstract
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.
Footnotes
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↵2 The abbreviations used are: Zp, zona pellucida protein; NOBOX, newborn ovary homeobox protein; POU5f1, POU domain class 5 transcription factor 1; Gdf9, growth differentiation factor 9; POU, Pit-Oct-Unc family; EMSA, electrophoretic mobility shift assay; CAST, cyclic amplification of sequence target analysis; GST, glutathione S-transferase; NX, Nobox; NXHD, Nobox homeodomain; NXNT, Nobox N terminus; NXCT, Nobox C terminus; HEK, human embryonic kidney; ChIP, chromatin immunoprecipitation; NBE, NOBOX DNA binding element; mNBE, mutant NOBOX DNA binding element; GAL4DBD, GAL4 DNA binding domain; UAS, upstream activation sequence; PGC, primordial germ cell; DE, distal enhancer; PE, proximal enhancer; TNAP, Tissue non-specific alkaline phosphatase.
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↵* 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.
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- Received April 26, 2006.
- Revision received September 13, 2006.
- The American Society for Biochemistry and Molecular Biology, Inc.
