Transcriptional regulation of the human reduced folate carrier promoter C: synergistic transactivation by Sp1 and C/EBP β and identification of a downstream repressor

The human reduced folate carrier (hRFC) is ubiquitously but differentially expressed in human tissues and its levels are regulated by up to six alternatively spliced non-coding regions (designated A1/A2, A, B, C, D, and E) and by at least four promoters. By transient transfections of HepG2 human hepatoma cells with 5′ and 3′ deletion constructs spanning 2883 bp of upstream sequence, a transcriptionally important region was localized to within 177 bp flanking the transcriptional start sites for exon C. By gel shift and chromatin immunoprecipitation assays, Sp1 and C/EBP β transcription factors were found to bind consensus elements (GC-box, CCAAT-box) within this region. The functional importance of these elements was confirmed by transient tranfections of HepG2 cells with hRFC-C reporter constructs in which these elements were mutated, and by co-transfections of Drosophila SL-2 cells with wild-type hRFC-C promoter and expression constructs for Sp1 and C/EBP β. Whereas both Sp1 and C/EBP β transactivated hRFC-C promoter activity, C/EBP α and γ were transcriptionally inert. Sp1 combined with C/EBP β resulted in a synergistic transactivation. In HepG2 cells, transfections with Sp1 and C/EBP β both increased endogenous levels of hRFC-C transcripts. By 3′ deletion analysis, a repressor sequence was localized to within 71 bp flanking the minimal promoter. On gel shifts, a novel transcriptional repressor was localized to within 30 bp. Collectively, these results identify transcriptionally important regions in the hRFC-C minimal promoter that include a GC-box and CCAAT-box, and suggest that cooperative interactions between Sp1 and C/EBP β are essential for hRFC-C transactivation. Another possible factor in the tissue-specific regulation of the hRFC-C region involves the downstream repressor flanking the minimal promoter.

人还原型叶酸载体（hRFC）在人体组织中普遍存在但表达有差异，其水平受多达六个可变剪接的非编码区域（分别命名为A1/A2、A、B、C、D和E）以及至少四个启动子的调控。通过用跨越2883bp上游序列的5′和3′缺失构建体对HepG2人肝癌细胞进行瞬时转染，一个具有转录重要性的区域被定位在紧邻外显子C转录起始位点的177bp范围内。通过凝胶迁移和染色质免疫沉淀分析，发现Sp1和C/EBPβ转录因子结合该区域内的共有元件（GC盒、CCAAT盒）。通过用这些元件发生突变的hRFC - C报告基因构建体对HepG2细胞进行瞬时转染，以及用野生型hRFC - C启动子和Sp1及C/EBPβ表达构建体对果蝇SL - 2细胞进行共转染，证实了这些元件的功能重要性。虽然Sp1和C/EBPβ都能反式激活hRFC - C启动子活性，但C/EBPα和γ在转录上无活性。Sp1与C/EBPβ结合产生协同的反式激活作用。在HepG2细胞中，用Sp1和C/EBPβ转染都增加了hRFC - C转录本的内源性水平。通过3′缺失分析，一个抑制序列被定位在紧邻最小启动子的71bp范围内。在凝胶迁移实验中，一种新的转录抑制因子被定位在30bp范围内。总之，这些结果确定了hRFC - C最小启动子中具有转录重要性的区域，包括一个GC盒和CCAAT盒，并表明Sp1和C/EBPβ之间的协同作用对hRFC - C的反式激活至关重要。hRFC - C区域组织特异性调控的另一个可能因素涉及紧邻最小启动子的下游抑制序列。