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

1.人α-叶酸受体(HFR)基因特异表达的核DNA结合蛋白的鉴定：我们已经鉴定出两个启动子，它们以组织特异性的方式独立地调节α-HFR基因的表达。为了进一步研究这些启动子的调控，我们通过启动子序列分析、凝胶漂移实验、核连续实验和核糖核酸酶保护实验研究了位于外显子1上游的启动子所涉及的转录元件。与在生理浓度叶酸中生长的wtKB细胞相比，我们发现(1)02细胞表达2%的HFR蛋白及其基因，(2)02细胞中HFR基因的转录速率比wtKB细胞低7倍，(3)核蛋白(S)在wtKB细胞中与转录起始点上游约400bp的DNA片段形成复合体，在C2细胞中显著降低；(4)基于多个人工合成的寡核苷酸序列的竞争性凝胶位移分析，发现-4391-452DNA片段含有该核蛋白(S)的特异结合部位。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。利用针对这些蛋白的抗体进行超移位分析，以表征该因子(S)对该区域的作用。核因子(S)的组织特异性表达：在未检测到αHFR表达的MCF-7、MDCK、ACHN、SN12C和TK-10细胞系中，核蛋白(S)与该DNA片段的结合也减少。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，并参与了抗叶酸抗性KB细胞受体表达的调节。

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.