STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF THE PROMOTER REGIONS OF THE NFKB2 GENE
STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF THE PROMOTER REGIONS OF THE NFKB2 GENE
复制标题
DOI：
10.1093/nar/23.12.2328
复制
发表时间：
1995-06-25
影响因子：
14.9
通讯作者：
NERI, A
中科院分区：
文献类型：
--
作者：
LOMBARDI, L;CIANA, P;NERI, A
为了阐明NFKB2基因（lyt - 10，NF - κBp100）的转录调控，我们对其启动子区域的结构和功能进行了表征。基于cDNA克隆的核苷酸序列以及NFKB2基因的5'侧翼基因组区域，在多个人类细胞系中进行的RT - PCR分析表明存在两个可选择的非编码第一外显子（Ia和Ib）。通过核糖核酸酶保护分析显示，在每个外显子上游鉴定出两个不同的启动子区域P1和P2，它们包含多个转录起始位点。对这些区域的序列分析表明，在外显子Ia上游有一个CAAT盒，并且在P1和P2内都有高GC含量区域。在每个外显子上游都发现了转录因子的共有结合位点，包括SP1、AP1和假定的NF - κB（κB位点）。特别是，鉴定出了六个κB位点，除了一个位点外，其余位点都能够在体外结合NF - κB复合物。将含有与氯霉素乙酰转移酶报告基因相连的P1和P2序列的质粒转染到HeLa细胞中，结果表明P1和P2都可以独立作为启动子发挥作用。将NF - κB效应质粒（NF - κBp5a和RelA）与连接到P1和P2的报告基因共转染，结果表明NFKB2启动子区域受NF - κB因子调控。RelA以剂量依赖的方式激活NFKB2启动子，而NF - κBp52则起抑制作用，这表明NFKB2基因可能受负反馈调节回路的控制。
In order to clarify the transcriptional regulation of the NFKB2 gene (lyt-10, NF-kappa Bp100), we have characterized the structure and function of its promoter regions. Based on the nucleotide sequence of cDNA clones and the 5' flanking genomic region of the NFKB2 gene, RT-PCR analysis in a number of human cell lines demonstrated the presence of two alternative noncoding first exons (Ia and Ib). Two distinct promoter regions, P1 and P2, were identified upstream of each exon, containing multiple sites of transcription initiation, as shown by RNase protection analysis. Sequence analysis of these regions showed a CAAT box upstream of exon Ia and high G-C content regions within both P1 and P2. Consensus binding sites for transcription factors, including SP1, API and putative NF-kappa B (kappa B sites), were found upstream of each exon. In particular, six kappa B sites were identified, all but one of them capable of binding NF-kappa B complexes in vitro. Transfection in HeLa cells of plasmids containing PI and P2 sequences linked to a chloramphenicol acetyltransferase reporter gene indicated that both P1 and P2 can act independently as promoters. Co-transfection of NF-kappa B effector plasmids (NF-kappa Bp5a and RelA) with a reporter gene linked to P1 and P2 showed that the NFKB2 promoter regions are regulated by NF-kappa B factors. RelA transactivates the NFKB2 promoter in a dose-dependent manner, whereas NF-kappa Bp52 acts as a repressor, indicating that the NFKB2 gene may be under the control of a negative feedback regulatory circuit.