Abstract
Expression of the rabbit uteroglobin gene is hormonally induced in cells of the endometrial epithelium during the preimplantation phase of pregnancy. Here we show that progesterone activation of the gene is mediated by two clusters of hormone responsive elements located between 2.4 and 2.7 kilobase pairs upstream of the transcriptional start site. Between these two clusters, genomic footprinting studies in the intact endometrial epithelium reveal the hormone-inducible occupancy of several cis-acting elements. One of the protected elements shows sequence homology to the consensus binding site of the transcription factor NF-Y, which binds to the element in gel shift experiments. This uteroglobin Y box is essential for enhancer activity in transient transfection experiments with endometrial and non-endometrial cell lines, in accordance with the ubiquitous expression of NF-Y. To understand why binding of this ubiquitous factor to the uteroglobin Y box in endometrium depends on hormone induction, we examined the chromatin structure of the relevant gene region. In the uninduced state, the enhancer region appears to be organized into positioned nucleosomes. Upon hormone induction, this nucleosomal pattern is lost and the enhancer region becomes hypersensitive to nucleases, suggesting that a hormone-induced change in the local chromatin structure unmasks previously unaccessible binding sites for transcription factors. Our results emphasize the limitations of using transient transfection assays for the functional analysis of cis-acting elements and underline the need for including the native chromatin organization in this kind of studies.
Highlights
Uteroglobin is a small globular protein originally described as the main protein component in the uterine secretion of rabbits in the preimplantation phase of pregnancy [1, 2]
We focused on the upstream region of the uteroglobin gene between Ϫ2.7 and Ϫ2.4 kb that encompasses two clusters of non-canonical putative progesterone responsive elements (PRE) and was previously shown to exhibit characteristic steroid hormone inducible and tissue-specific DNase I-hypersensitive sites [17] (Fig. 1A)
Cell nuclei from the endometrial epithelium and liver were treated with DNase I and the digested DNA was analyzed by LM-PCR genomic footprinting [22, 23, 39]
Summary
Animals and Treatments—Adult 1/2 to 1-year-old female rabbits (New Zealand White or Chinchilla-Bastard, 3– 4 kg) were housed in individual cages under controlled conditions of temperature and light (12 h light-dark) and kept separated from male rabbits to prevent pheromone-triggered ovulation. The uteroglobin gene fragments were cut from the plasmid, purified by gel electrophoresis, and 32P-labeled by Klenow filling-in reaction. The cells were maintained in Dulbecco’s modified EagleЈs medium (Life Technologies, Inc.) supplemented with 10% (Ishikawa) or 4% (RBE-7) fetal calf serum (c.c. pro GmbH, Germany), penicillin (100 IU/ml), and streptomycin (100 g/ml) and cultivated at 37 °C (Ishikawa) or 33 °C (RBE-7) and 5% CO2. After transfection the cells were washed with phosphate-buffered saline and the medium changed for phenol red-free DMEM (Life Technologies, Inc.) supplemented with 5% fetal calf serum treated with charcoal dextran [32]. Mapping of Nucleosome and Nuclease Hypersensitive Sites—Digestion of cell nuclei with methidiumpropyl-EDTA-FeII (MPE), separation of the DNA fragments in agarose gels, blotting, and indirect end labeling [36, 37] were performed as described previously [22]. Quantitation of Radiolabeled DNA—Quantitative evaluation was performed directly from the dried gel using a PhosphorImager and ImageQuant software (Molecular Dynamics Inc., Sunnyvale, CA) or from autoradiographs using a laser scanner
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