Lee C S, Oka T
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1992 Mar 25;267(9):5797-801.
The sequence-specific binding pattern of mammary nuclear proteins to the 545-base pair (bp) 5'-flanking region of the mouse beta-casein gene was compared between pregnant and lactating mice by a gel mobility shift assay. By using appropriate probes, two complexes were detected only during pregnancy, whereas an additional four complexes were detected during both pregnancy and lactation. The two pregnancy-specific complexes showed identical electrophoretic mobility and were cross-competed with two unlabeled DNA fragments used to generate the probes. Methylation interference experiments indicated that the two binding regions involved guanosine residues at nucleotides -350 and -8, and the sequences around each guanosine residue have a common palindromic sequence, 5'-TGAT/ATCA-3'. This binding factor is termed pregnancy-specific mammary nuclear factor. In gene transfection experiments, expression of the chimeric beta-casein-CAT gene linked to the 545 bp of mouse beta-casein gene promoter region was maximally induced when transfected mammary epithelial cells were cultured with the lactogenic hormones prolactin, hydrocortisone, and insulin, whereas it was very low when insulin alone was added. The addition of progesterone together with the lactogenic hormones inhibited the hormonal induction of the chimeric gene, whereas co-transfection with an oligonucleotide containing the pregnancy-specific mammary nuclear factor binding site substantially overcome the progesterone-mediated repression of transcription. Furthermore, mutation of the pregnancy-specific mammary nuclear factor binding sites of the chimeric beta-casein-CAT gene resulted in attenuation of the inhibitory effect of progesterone without blocking the stimulatory effect of the lactogenic hormones. These results indicate the presence of a pregnancy-specific mammary nuclear factor(s) that bind to two separate sites of the beta-casein gene promoter and suggest that it may serve as a repressor that mediates the inhibitory action of progesterone on beta-casein gene transcription.
通过凝胶迁移率变动分析，比较了妊娠小鼠和泌乳小鼠乳腺核蛋白与小鼠β-酪蛋白基因545碱基对（bp）5'-侧翼区域的序列特异性结合模式。使用合适的探针，仅在妊娠期间检测到两种复合物，而在妊娠和泌乳期间均检测到另外四种复合物。这两种妊娠特异性复合物显示出相同的电泳迁移率，并与用于生成探针的两个未标记DNA片段发生交叉竞争。甲基化干扰实验表明，这两个结合区域涉及核苷酸-350和-8处的鸟苷残基，并且每个鸟苷残基周围的序列具有共同的回文序列5'-TGAT/ATCA-3'。这种结合因子被称为妊娠特异性乳腺核因子。在基因转染实验中，当用催乳素、氢化可的松和胰岛素等泌乳激素培养转染的乳腺上皮细胞时，与小鼠β-酪蛋白基因启动子区域545 bp相连的嵌合β-酪蛋白-CAT基因的表达被最大程度地诱导，而仅添加胰岛素时表达非常低。孕酮与泌乳激素一起添加会抑制嵌合基因的激素诱导，而与含有妊娠特异性乳腺核因子结合位点的寡核苷酸共转染可基本克服孕酮介导的转录抑制。此外，嵌合β-酪蛋白-CAT基因的妊娠特异性乳腺核因子结合位点的突变导致孕酮抑制作用的减弱，而不阻断泌乳激素的刺激作用。这些结果表明存在一种妊娠特异性乳腺核因子，它与β-酪蛋白基因启动子的两个不同位点结合，并表明它可能作为一种阻遏物，介导孕酮对β-酪蛋白基因转录的抑制作用。
