Lombardi L, Ciana P, Cappellini C, Trecca D, Guerrini L, Migliazza A, Maiolo A T, Neri A
Laboratorio di Ematologia Sperimentale e Genetica Molecolare, Università di Milano, Ospedale Maggiore IRCCS, Italy.
Nucleic Acids Res. 1995 Jun 25;23(12):2328-36. doi: 10.1093/nar/23.12.2328.
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 (1a and 1b). 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 1a and high G-C content regions within both P1 and P2. Consensus binding sites for transcription factors, including SP1, AP1 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 P1 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 Bp52 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.
为了阐明NFKB2基因（lyt-10，NF-κBp100）的转录调控机制，我们对其启动子区域的结构和功能进行了表征。基于NFKB2基因的cDNA克隆核苷酸序列及其5'侧翼基因组区域，在多种人类细胞系中进行的RT-PCR分析表明存在两个可变的非编码第一外显子（1a和1b）。在每个外显子上游鉴定出两个不同的启动子区域P1和P2，如核糖核酸酶保护分析所示，它们含有多个转录起始位点。对这些区域的序列分析显示，在1a外显子上游有一个CAAT框，且P1和P2内均有高G-C含量区域。在每个外显子上游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）。特别地，鉴定出六个κB位点，其中除一个外，其余均能够在体外结合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序列的质粒转染至HeLa细胞表明，P1和P2均可独立作为启动子发挥作用。将NF-κB效应质粒（NF-κBp52和RelA）与与P1和P2相连的报告基因共转染表明，NFKB2启动子区域受NF-κB因子调控。RelA以剂量依赖性方式反式激活NFKB2启动子，而NF-κBp52则作为阻遏物发挥作用，这表明NFKB2基因可能受负反馈调节回路的控制。
