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Identification and cDNA cloning of the transcriptional factors regulating the tissue-specific expression of human alpha-folate receptor gene

调节人α-叶酸受体基因组织特异性表达的转录因子的鉴定及cDNA克隆

基本信息

批准号：
09670793
负责人：
SAIKAWA Yutaka
金额：
$ 1.86万
依托单位：
Kanazawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

1. I-dentification of the nuclear DNA-binding proteins involving the specific expression of alpha human folate receptor (hFR) gene : we have identified two promoters which are independently active in a tissue-specific manner in regulation of the alpha hFR gene expression. To further characterize the regulation of these promoters, the transcriptional elements involved in the promoter located upstream of exon 1 were investigated in the transport-defective methotrexate-resistant KB cells (02) by sequence analysis of the promoter, gel shift assays, nuclear run-on assays, and RNase protection assays. Compared to wtKB cells grown in physiologic concentrations of folate, we demonstrate that (1) the 02 cells expressed 2% of a hFR protein and its mRNA ; (2) the transcription rate of a hFR gene was reduced 7 fold in the 02 cells relative to wtKB cells ; (3) the nuclear protein(s) that forms a complex with a -352/-461 (109 bp) DNA fragment located approximately 400 bp upstream of the transcription start site was demonstrated in wtKB cells and was significantly reduced in the C2 cells ; (4) based on competitive gel shift assays using several synthetic oligonucleotides corresponding - 426/-461 DNA sequences, a -4391-452 DNA fragment contained the specific binding sites of this nuclear protein(s). This sequence contains potential binding sites of CCAAT-enhancer binding protein beta, GATA-2, and Ets-1. The supershift assays using antibodies against these proteins will be performed to characterize the factor(s) to this region.2. Tissue specific expression of the nuclear factor(s) : the binding of nuclear protein(s) to this DNAfragment was also decreased in MCF-7, MDCK, ACHN, SN12C, and TK- 10 celllines in which expression of the alpha hFR is not detectable. These results suggest that this factor may be involved in the tissue-specific expression of alpha hFR gene and in the modulation of receptor expression in antifolate resistant KB cells.

1.鉴定与α-人叶酸受体（hFR）基因特异性表达有关的核DNA结合蛋白：我们鉴定了两种启动子，它们以组织特异性方式独立地调节α-hFR基因的表达。为了进一步表征这些启动子的调控，通过启动子的序列分析、凝胶迁移试验、核运行试验和RNA酶保护试验，在转运缺陷型甲氨蝶呤耐药KB细胞（02）中研究了位于外显子1上游的启动子中涉及的转录元件。与在生理浓度的叶酸盐中生长的wtKB细胞相比，我们证明：（1）02细胞表达2%的hFR蛋白及其mRNA ;（2）相对于wtKB细胞，02细胞中hFR基因的转录速率降低7倍;（3）与-352/-461（109 bp）形成复合物的核蛋白在转录起始位点上游约400 bp处的DNA片段在wtKB细胞中被证实，并且在C2细胞中显著减少;（4）用几种人工合成的寡核苷酸对-426/-461DNA序列进行竞争性凝胶迁移分析，发现-4391-452DNA片段含有该核蛋白的特异性结合位点。该序列包含CCAAT增强子结合蛋白β、加塔-2和Ets-1的潜在结合位点。将使用针对这些蛋白质的抗体进行超位移测定，以表征该区域的因子。核因子的组织特异性表达：在MCF-7、MDCK、ACHN、SN 12 C和TK- 10细胞系中，核蛋白与该DNA片段的结合也降低，其中未检测到α hFR的表达。这些结果表明，该因子可能参与了α hFR基因的组织特异性表达，并参与了抗叶酸剂耐药KB细胞中受体表达的调节。