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

1. i -鉴定涉及α人叶酸受体（hFR）基因特异性表达的核dna结合蛋白：我们已经鉴定了两个启动子，它们以组织特异性的方式独立地调控α人叶酸受体基因表达。为了进一步表征这些启动子的调控，在转运缺陷的抗甲氨蝶呤KB细胞（02）中，通过启动子序列分析、凝胶移位试验、核运行试验和RNase保护试验，研究了位于外显子1上游的启动子所涉及的转录元件。与在叶酸生理浓度下生长的wtKB细胞相比，我们发现(1)02细胞表达了2%的hFR蛋白及其mRNA；(2)与wtKB细胞相比，hFR基因在02细胞中的转录率降低了7倍；(3)与转录起始位点上游约400 bp的-352/-461 (109 bp) DNA片段形成复合物的核蛋白(s)在wtKB细胞中发现，在C2细胞中显著减少；(4)利用几种合成的与- 426/-461 DNA序列相对应的寡核苷酸进行竞争性凝胶转移分析，发现-4391-452 DNA片段包含该核蛋白的特定结合位点。该序列包含ccaat增强子结合蛋白β、GATA-2和Ets-1的潜在结合位点。使用针对这些蛋白质的抗体进行超移测定，以表征该区域的因子。核因子的组织特异性表达：在没有检测到α - hFR表达的MCF-7、MDCK、ACHN、SN12C和TK- 10细胞系中，核蛋白与该dna片段的结合也减少了。这些结果表明，该因子可能参与了抗叶酸抗性KB细胞中α - hFR基因的组织特异性表达和受体表达的调节。

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.