Pan Yuan-Xiang, Chen Hong, Thiaville Michelle M, Kilberg Michael S
Department of Biochemistry and Molecular Biology, Genetics Institute, Shands Cancer Center and Center for Nutritional Sciences, University of Florida College of Medicine, Gainesville, Florida 32610, USA.
Biochem J. 2007 Jan 1;401(1):299-307. doi: 10.1042/BJ20061261.
Expression of ATF3 (activating transcription factor 3) is induced by a variety of environmental stress conditions, including nutrient limitation. In the present study, we demonstrate that the increase in ATF3 mRNA content following amino acid limitation of human HepG2 hepatoma cells is dependent on transcriptional activation of the ATF3 gene, through a highly co-ordinated amino acid-responsive programme of transcription factor synthesis and action. Studies using transient over-expression and knockout fibroblasts showed that several ATF and C/EBP (CCAAT/enhancer-binding protein) family members contribute to ATF3 regulation. Promoter analysis showed that a C/EBP-ATF composite site at -23 to -15 bp relative to the transcription start site of the ATF3 gene functions as an AARE (amino acid response element). Chromatin immunoprecipitation demonstrated that amino acid limitation increased ATF4, ATF3, and C/EBPbeta binding to the ATF3 promoter, but the kinetics of each was markedly different. Immediately following histidine removal, there was a rapid increase in histone H3 acetylation prior to an enhancement in ATF4 binding and in histone H4 acetylation. These latter changes closely paralleled the initial increase in RNA pol II (RNA polymerase II) binding to the promoter and in the transcription rate from the ATF3 gene. The increase in ATF3 and C/EBPbeta binding was considerably slower and more closely correlated with a decline in transcription rate. A comparison of the recruitment patterns between ATF and C/EBP transcription factors and RNA polymerase II at the AARE of several amino acid-responsive genes revealed that a highly co-ordinated response programme controls the transcriptional activation of these genes following amino acid limitation.
激活转录因子3（ATF3）的表达可由多种环境应激条件诱导，包括营养限制。在本研究中，我们证明了人类HepG2肝癌细胞在氨基酸限制后ATF3 mRNA含量的增加依赖于ATF3基因的转录激活，这是通过转录因子合成和作用的高度协调的氨基酸反应程序实现的。使用瞬时过表达和基因敲除成纤维细胞的研究表明，几个ATF和CCAAT增强子结合蛋白（C/EBP）家族成员参与了ATF3的调控。启动子分析表明，相对于ATF3基因转录起始位点-23至-15 bp处的C/EBP-ATF复合位点作为氨基酸反应元件（AARE）发挥作用。染色质免疫沉淀表明，氨基酸限制增加了ATF4、ATF3和C/EBPβ与ATF3启动子的结合，但每种结合的动力学明显不同。在去除组氨酸后，立即观察到组蛋白H3乙酰化迅速增加，随后ATF4结合增强以及组蛋白H4乙酰化增加。这些后期变化与RNA聚合酶II（RNA pol II）与启动子结合的初始增加以及ATF3基因转录速率的增加密切平行。ATF3和C/EBPβ结合的增加相当缓慢，并且与转录速率的下降更密切相关。对几个氨基酸反应基因的AARE处ATF和C/EBP转录因子与RNA聚合酶II的募集模式进行比较，结果显示，高度协调的反应程序控制了氨基酸限制后这些基因的转录激活。
