Erb M J, Camacho D, Xie W, Maslikowski B M, Fielding B, Ghosh R, Poujade F-A, Athar M, Assee S, Mantella L-E, Bédard P-A
Department of Biology, McMaster University, Hamilton, Ontario, Canada.
Department of Biology, McMaster University, Hamilton, Ontario, Canada
Mol Cell Biol. 2016 Nov 14;36(23):2890-2902. doi: 10.1128/MCB.00338-16. Print 2016 Dec 1.
The activation of the growth arrest-specific (gas) p20K gene depends on the interaction of C/EBPβ with two elements of a 48-bp promoter region termed the quiescence-responsive unit (QRU). Here we identify extracellular signal-related kinase 2 (ERK2) as a transcriptional repressor of the p20K QRU in cycling chicken embryo fibroblasts (CEF). ERK2 binds to repeated GAAAG sequences overlapping the C/EBPβ sites of the QRU. The recruitment of ERK2 and C/EBPβ is mutually exclusive and dictates the expression of p20K. C/EBP homologous protein (CHOP) was associated with C/EBPβ under conditions promoting endoplasmic reticulum (ER) stress and, to a lesser extent, in cycling CEF but was not detectable when C/EBPβ was immunoprecipitated from contact-inhibited cells. During ER stress, overexpression of CHOP inhibited p20K, while its downregulation promoted p20K, indicating that CHOP is also a potent inhibitor of p20K. Transcriptome analyses revealed that hypoxia-responsive genes are strongly induced in contact-inhibited but not serum-starved CEF, and elevated levels of nitroreductase activity, a marker of hypoxia, were detected at confluence. Conditions of hypoxia (2% O) induced growth arrest in subconfluent CEF and markedly stimulated p20K expression, suggesting that the control of proliferation and gas gene expression is closely linked to limiting oxygen concentrations associated with high cell densities.
生长停滞特异性（gas）p20K基因的激活取决于C/EBPβ与一个48bp启动子区域的两个元件（称为静止反应单元，QRU）的相互作用。在此，我们确定细胞外信号相关激酶2（ERK2）是鸡胚成纤维细胞（CEF）中p20K QRU的转录抑制因子。ERK2与重叠于QRU的C/EBPβ位点的重复GAAAG序列结合。ERK2和C/EBPβ的募集相互排斥，并决定了p20K的表达。在促进内质网（ER）应激的条件下，C/EBP同源蛋白（CHOP）与C/EBPβ相关，在一定程度上也与处于增殖状态的CEF相关，但当从接触抑制的细胞中免疫沉淀C/EBPβ时，未检测到CHOP。在内质网应激期间，CHOP的过表达抑制p20K，而其下调则促进p20K，这表明CHOP也是p20K的有效抑制剂。转录组分析显示，缺氧反应基因在接触抑制而非血清饥饿的CEF中被强烈诱导，并且在汇合时检测到作为缺氧标志物的硝基还原酶活性水平升高。缺氧条件（2%O）在亚汇合的CEF中诱导生长停滞并显著刺激p20K表达，这表明增殖控制和gas基因表达与与高细胞密度相关的有限氧浓度密切相关。
