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.
Highlights
Health and a March of Dimes Basil O’Connor Award (5-FY02–266)
It is unknown whether NOBOX directly or indirectly regulates transcription of these oocyte-specific genes during development of the ovarian follicle
We have identified a consensus sequence, TAATTG, as NOBOX DNA binding elements using purified proteins in a random DNA selection assay (Fig. 1)
Summary
Expression and Purification of GST-NXHD—To create the pET41b-Nobox homeodomain protein (GST-NXHD) and bacterial expression construct, the insert was amplified by PCR using primers containing EcoRI and HindIII sites, and cloned into pET41b-EcoRI/HindIII (Novagen, Madison, WI). 0.5 pmol of short double-stranded DNA containing a 15-bp random sequence flanked by 20-bp fixed sequences, CAST oligonucleotides (Table 1), was incubated with 100 ng of purified GST-NXHD protein for 20 min at room temperature. The fixed ovaries were washed with cold 1ϫ phosphatebuffered saline two times, homogenized into 4 ml of homogenization buffer (50 mM sodium phosphate buffer, pH 7.5, 10% glycerol, 10 mM -mercaptoethanol, 50 mM phenylmethylsulfonyl fluoride, and protein inhibitors) and prepared for immunoprecipitation using the modified protocol of ChIP assay kit (Upstate Biotechnology Inc., Lake Placid, NY). The samples were pre-cleared with salmon sperm DNA/protein A-agarose (Upstate Biotechnology Inc.) and incubated with either 10 l of goat anti-Nobox antibody or IgG at 4 °C overnight. After 33 cycles of amplification (all primer sets), PCR products were run on 2% agarose gels and analyzed
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