Chen Chin, Dudenhausen Elizabeth, Chen Hong, Pan Yuan-Xiang, Gjymishka Altin, Kilberg Michael S
Department of Biochemistry and Molecular Biology, Genetics Institute, and Shands Cancer Center, University of Florida College of Medicine, Gainesville, FL 32610, USA.
Biochem J. 2005 Nov 1;391(Pt 3):649-58. doi: 10.1042/BJ20050882.
For animals, dietary protein is critical for the nutrition of the organism and, at the cellular level, protein nutrition translates into amino acid availability. Amino acid deprivation triggers the AAR (amino acid response) pathway, which causes enhanced transcription from specific target genes. The present results show that C/EBPbeta (CCAAT/enhancer-binding protein beta) mRNA and protein content were increased following the deprivation of HepG2 human hepatoma cells of a single amino acid. Although there was a modest increase in mRNA half-life following histidine limitation, the primary mechanism for the elevated steady-state mRNA was increased transcription. Transient transfection documented that C/EBPbeta genomic fragments containing the 8451 bp 5' upstream of the transcription start site did not contain amino-acid-responsive elements. However, deletion analysis of the genomic region located 3' downstream of the protein coding sequence revealed that a 93 bp fragment contained an amino-acid-responsive activity that functioned as an enhancer. Exogenous expression of ATF4 (activating transcription factor 4), known to activate other genes through amino acid response elements, caused increased transcription from reporter constructs containing the C/EBPbeta enhancer in cells maintained in complete amino acid medium. Chromatin immunoprecipitation demonstrated that RNA polymerase II is bound at the C/EBPbeta promoter and at the 93 bp regulatory region in vivo, whereas ATF4 binds to the enhancer region only. Immediately following amino acid removal, the kinetics of binding for ATF4, ATF3, and C/EBPbeta itself to the 93 bp regulatory region were similar to those observed for the amino-acid-responsive asparagine synthetase gene. Collectively the findings show that expression of C/EBPbeta, which contributes to the regulation of amino-acid-responsive genes, is itself controlled by amino acid availability through transcription.
对于动物而言，膳食蛋白质对机体营养至关重要，在细胞水平上，蛋白质营养转化为氨基酸的可利用性。氨基酸剥夺会触发氨基酸应答（AAR）途径，该途径会导致特定靶基因的转录增强。目前的结果表明，在人肝癌HepG2细胞缺乏单一氨基酸后，C/EBPβ（CCAAT/增强子结合蛋白β）的mRNA和蛋白质含量增加。尽管在组氨酸限制后mRNA半衰期有适度增加，但稳态mRNA升高的主要机制是转录增加。瞬时转染表明，包含转录起始位点上游8451 bp 5'的C/EBPβ基因组片段不包含氨基酸应答元件。然而，对位于蛋白质编码序列下游3'的基因组区域进行缺失分析发现，一个93 bp的片段具有作为增强子发挥作用的氨基酸应答活性。已知通过氨基酸应答元件激活其他基因的ATF4（激活转录因子4）的外源表达，在完全氨基酸培养基中培养的细胞中，导致含有C/EBPβ增强子的报告构建体的转录增加。染色质免疫沉淀表明，RNA聚合酶II在体内与C/EBPβ启动子和93 bp调控区域结合，而ATF4仅与增强子区域结合。在去除氨基酸后，ATF4、ATF3和C/EBPβ自身与93 bp调控区域结合的动力学与氨基酸应答的天冬酰胺合成酶基因所观察到的相似。这些发现共同表明，对氨基酸应答基因调控有贡献的C/EBPβ的表达本身通过转录受氨基酸可利用性的控制。
