Kasinski Andrea, Dong Xueyuan, Khuri Fadlo R, Boss Jeremy, Fu Haian
Program in Genetics and Molecular Biology, Emory University, Atlanta, Georgia, United States of America; Department of Pharmacology, Emory University, Atlanta, Georgia, United States of America.
Department of Pharmacology, Emory University, Atlanta, Georgia, United States of America.
PLoS One. 2014 Apr 1;9(4):e93480. doi: 10.1371/journal.pone.0093480. eCollection 2014.
Aberrant expression of oncogenic 14-3-3 proteins is correlated with poor survival of cancer patients. While the underlying mechanism of the abnormal expression in tumors remains elusive for the six oncogenic 14-3-3 isoforms; the potential involvement of a transcriptional component has been suggested. Unfortunately, little experimental data has been reported to support this hypothesis. In this study we describe the genetic structure of YWHAZ, the gene encoding 14-3-3ζ, including the identification of previously unreported transcript variants. In total, five transcript variants were revealed and their expressions confirmed in a panel of cell lines. Expressed sequence tag (EST) database mining and in vitro rapid-amplification of cDNA ends (RACE) confirmed that one variant, 1c, represents >80% of the expressed transcripts, which is also the most efficiently translated. An analysis of the proximal promoter of this variant revealed a functional Cyclic-AMP Response Element (CRE). Factors that bound to the CRE element were recognized through fractionation and subsequent EMSAs. This analysis identified CREB and ATF-1 as the trans-interacting factors. Cell-based assays confirm that ATF-1, and to a lesser extent CREB, bind the endogenous YWHAZ promoter especially under TNF-α stimulating conditions. In support of a role of ATF-1 in the regulation of YWHAZ, silencing of ATF-1 resulted in a marked reduction in two of the five YWHAZ transcripts. These data suggest a novel mechanism for the transcriptional regulation of a major pro-survival gene, YWHAZ, by ATF-1.
致癌性14-3-3蛋白的异常表达与癌症患者的不良生存预后相关。虽然肿瘤中六种致癌性14-3-3亚型异常表达的潜在机制仍不清楚，但有研究表明转录成分可能参与其中。遗憾的是，几乎没有实验数据支持这一假设。在本研究中，我们描述了编码14-3-3ζ的YWHAZ基因的遗传结构，包括鉴定以前未报道的转录变体。总共发现了五种转录变体，并在一组细胞系中证实了它们的表达。通过表达序列标签（EST）数据库挖掘和体外cDNA末端快速扩增（RACE）证实，一种变体1c占表达转录本的80%以上，也是翻译效率最高的变体。对该变体近端启动子的分析揭示了一个功能性的环磷酸腺苷反应元件（CRE）。通过分级分离和随后的电泳迁移率变动分析（EMSA）识别与CRE元件结合的因子。该分析确定CREB和ATF-1为反式相互作用因子。基于细胞的分析证实，ATF-1，以及程度较轻的CREB，特别是在TNF-α刺激条件下与内源性YWHAZ启动子结合。为支持ATF-1在YWHAZ调控中的作用，沉默ATF-1导致五种YWHAZ转录本中的两种显著减少。这些数据提示了一种由ATF-1对主要促生存基因YWHAZ进行转录调控的新机制。
