Isolation of estrogen-responsive genes with a CpG island library
Isolation of estrogen-responsive genes with a CpG island library
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DOI：
10.1128/mcb.18.1.442
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发表时间：
1998-01-01
影响因子：
5.3
通讯作者：
Muramatsu, M
中科院分区：
文献类型：
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作者：
Watanabe, T;Inoue, S;Muramatsu, M
为了分离新的雌激素反应基因，我们利用一个富含调控区的CpG岛文库，采用基因组结合位点（GBS）克隆法筛选了能与重组雌激素受体蛋白结合的CpG岛，筛选出6个含有完全、不完全和/或多个半回文雌激素反应元件（ERes）的CpG岛。北方印迹分析表明，这些基因组片段均与特定的mRNA杂交，提示与这些ERE相关的基因可能在人细胞中转录。然后分别从人胎盘和人乳腺癌MCF-7细胞的文库中分离出与其中两个ERE相关的cDNA，即EB 1和EB 9。该增加被放线菌素D抑制，但不被放线菌酮抑制，表明上调不需要蛋白质合成。与EB 1相关的cDNA编码一个类似于细胞色素c氧化酶亚基VIIa的114个氨基酸的蛋白质，命名为COX 7 RP（细胞色素c氧化酶亚基VII相关蛋白），与EB 9相关的cDNA仅与表达序列标签同源，并被命名为EBAG 9（estrogen receptor-binding fragment-associated gene 9）。EB 1的回文ERE位于COX 7 RP的内含子中，EB 9的回文ERE位于cDNA的5'上游区。将这两个EREs插入鸡β-珠蛋白启动子的5'上游区域后，在氯霉素乙酰转移酶试验中均表现出明显的雌激素依赖性增强子活性。因此，我们建议，本文所述的从CpG岛文库中分离DNA结合位点的CPG-GBS方法将有助于某些转录因子的新靶基因的鉴定。
In order to isolate novel estrogen-responsive genes, we utilized a CpG island library in which the regulatory regions of genes are enriched, CpG islands were screened for the ability to bind to a recombinant estrogen receptor protein with a genomic binding site (GBS) cloning method, Six CpG islands were selected, and they contained perfect, imperfect, and/or multiple half-palindromic estrogen-responsive elements (EREs). Northern blot analysis of various human cells showed that all these genomic fragments hybridized to specific mRNAs, suggesting that the genes associated with these EREs might be transcribed in human cells, Then cDNAs associated with two of them, EB1 and EB9, were isolated from libraries of human placenta and MCF-7 cells derived from a human breast cancer, respectively, Both transcripts were increased by estrogen in MCF-7 cells, The increase is inhibited by actinomycin D but not by cycloheximide, indicating that no protein synthesis is required for the up-regulation, The cDNA associated with EB1 encodes a 114-amino-acid protein similar to the cytochrome c oxidase subunit VIIa, named COX7RP (cytochrome c oxidase subunit VII-related protein), The cDNA associated with EB9 is homologous only to an express sequence tag and was named EBAG9 (estrogen receptor-binding fragment-associated gene 9). The palindromic ERE of EB1 is located in an intron of COX7RP, and that of EB9 is in the 5' upstream region of the cDNA. Both EREs had significant estrogen-dependent enhancer activities in a chloramphenicol acetyltransferase assay, when they were inserted into the 5' upstream region of the chicken beta-globin promoter, We therefore propose that the CPG-GBS method described here for isolation of the DNA binding site from the CpG island library would be useful for identification of novel target genes of certain transcription factors.