Oztürk Arzu, Donald Lynda J, Li Lin, Duckworth Harry W, Duckworth Mary Lynn
Departments of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada R3E 3J7.
Endocrinology. 2006 Sep;147(9):4319-29. doi: 10.1210/en.2006-0492. Epub 2006 Jun 22.
The factors that regulate the developmental expression of the rodent prolactin gene family in placenta remain poorly defined. We previously identified an enhancer element in the 5' flanking region of one family member, rat placental lactogen II (rPLII), which could target reporter gene expression to the placenta in transgenic mice; this enhancer functioned in the Rcho rat trophoblast cell line but not in the rat pituitary GC cell line. In further experiments to identify the factors that bind this element, we have selectively enriched for DNA binding proteins in nuclear extract from Rcho cells using magnetic beads coupled to a 43-bp enhancer oligonucleotide. Tryptic peptides of bound proteins were analyzed by HPLC coupled off-line to matrix-assisted laser desorption ionization time of flight mass spectrometry. Several peptides of AP2 gamma, a key trophoblast cell-specific transcription factor, were identified. Gel mobility shift assays using AP2 gamma-specific antiserum and mutant enhancer oligonucleotides demonstrated binding specifically to the FP2 DNase I-protected region of the element, identifying an atypical binding site for this factor. In cotransfection assays in rat pituitary GC cells, AP2 gamma transactivated the enhancer via this region. Chromatin immunoprecipitation assays confirmed AP2 gamma occupancy of the enhancer region in situ in the nuclei of Rcho giant cells. These data support a role for AP2 gamma in the placental giant cell-specific expression of the rPLII gene and provide the first direct evidence for the involvement of a placental-specific transcription factor in the regulation of a member of this gene family.
调节啮齿动物催乳素基因家族在胎盘中发育性表达的因素仍不清楚。我们之前在一个家族成员大鼠胎盘催乳素II（rPLII）的5'侧翼区域鉴定了一个增强子元件，该元件可将报告基因的表达靶向转基因小鼠的胎盘；这种增强子在Rcho大鼠滋养层细胞系中起作用，但在大鼠垂体GC细胞系中不起作用。在进一步鉴定与该元件结合的因子的实验中，我们使用与43bp增强子寡核苷酸偶联的磁珠从Rcho细胞的核提取物中选择性富集DNA结合蛋白。通过与基质辅助激光解吸电离飞行时间质谱联用的HPLC分析结合蛋白的胰蛋白酶肽段。鉴定出了关键的滋养层细胞特异性转录因子AP2γ的几种肽段。使用AP2γ特异性抗血清和突变增强子寡核苷酸的凝胶迁移率变动分析表明，其特异性结合该元件的FP2 DNase I保护区域，确定了该因子的一个非典型结合位点。在大鼠垂体GC细胞的共转染实验中，AP2γ通过该区域反式激活增强子。染色质免疫沉淀分析证实了AP2γ在Rcho巨细胞的细胞核中原位占据增强子区域。这些数据支持AP2γ在rPLII基因胎盘巨细胞特异性表达中的作用，并为胎盘特异性转录因子参与该基因家族成员的调控提供了首个直接证据。
