Choi Youngsok, Rajkovic Aleksandar
Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas 77030, USA.
J Biol Chem. 2006 Nov 24;281(47):35747-56. doi: 10.1074/jbc.M604008200. Epub 2006 Sep 22.
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.
Nobox（新生卵巢同源盒基因）缺陷会破坏小鼠早期卵泡发生以及卵母细胞特异性基因的表达。在此，我们通过序列靶向分析的循环扩增和突变分析，利用随机生成的寡核苷酸文库鉴定出了几个顺式作用位点TAATTG、TAGTTG和TAATTA作为NOBOX DNA结合元件（NBEs）。我们发现NOBOX以高亲和力优先结合到NOBOX结合元件上。此外，我们发现小鼠Pou5f1和Gdf9的启动子区域分别包含一个（-426）和三个NOBOX结合元件（-786、-967和-1259）。NOBOX以高亲和力结合到这些假定的NOBOX结合元件上，并增强了由含有NOBOX结合元件的小鼠Pou5f1和Gdf9启动子驱动的荧光素酶报告基因的转录活性。在染色质免疫沉淀试验中，来自Pou5f1和Gdf9启动子的DNA序列与抗NOBOX抗体共沉淀。这些结果表明，在早期卵泡发生过程中，NOBOX直接调控卵母细胞中Pou5f1和Gdf9的转录。
