Transcriptional Regulation of the TATA-Binding Protein by Ras Cellular Signaling

Our previous studies have demonstrated that the level of the central transcription factor TATA-binding protein (TBP) is increased in cells expressing the hepatitis B virus (HBV) X protein through the activation of the Ras signaling pathway, which serves to enhance both RNA polymerase I and III promoter activities. To understand the mechanism by which TBP is regulated, we have investigated whether enhanced expression is modulated at the transcriptional level. Nuclear run-on assays revealed that the HBV X protein increases the number of active transcription complexes on the TBP gene. In transient-transfection assays with both transformed and primary hepatocytes, the human TBP promoter was shown to be induced by expression of the HBV X protein in a Ras-dependent manner, requiring both Ral guanine nucleotide dissociation stimulator (RalGDS) and Raf signaling. Transient overexpression of TBP did not affect TBP promoter activity. To further delineate the downstream Ras-mediated events contributing to TBP promoter regulation in primary rat hepatocytes, the best-characterized Ras effectors, Raf, phosphoinositide 3-kinase (PI-3 kinase), and RalGDS, were examined. Activation of either Raf or RalGDS, but not that of PI-3 kinase, was sufficient to induce TBP promoter activity. Both Raf- and RalGDS-mediated induction required the activation of mitogen-activated protein kinase kinase (MEK). In addition, another distinct Ras-activated pathway, which does not require MEK activation, appears to induce TBP promoter activity. Analysis of the DNA sequence requirement within the TBP promoter responsible for these regulatory events defined three distinct regions that modulate the abilities of Raf, RalGDS, and the Ras-dependent, MEK-independent pathways to regulate human TBP promoter activity. Together, these results provide new evidence that TBP can be regulated at the transcriptional level and identify three distinct Ras-activated pathways that modulate this central eukaryotic transcription factor.

我们前期研究已证实，在表达乙型肝炎病毒（hepatitis B virus, HBV）X蛋白的细胞中，核心转录因子TATA盒结合蛋白（TATA-binding protein, TBP）的水平通过Ras信号通路的激活而升高，该效应可增强RNA聚合酶Ⅰ和Ⅲ的启动子活性。为阐明TBP的调控机制，我们探究了其表达上调是否在转录水平受到调控。核连缀转录分析结果显示，HBV X蛋白可增加TBP基因上活性转录复合物的数量。在转化肝细胞与原代肝细胞的瞬时转染实验中，研究发现HBV X蛋白可通过Ras依赖的方式诱导人TBP启动子的活性，且该过程需要Ral鸟苷酸解离刺激因子（Ral guanine nucleotide dissociation stimulator, RalGDS）与Raf信号的共同参与。TBP的瞬时过表达并不会影响TBP启动子的活性。为进一步明确原代大鼠肝细胞中介导TBP启动子调控的下游Ras相关事件，我们对目前研究最为充分的Ras效应因子——Raf、磷脂酰肌醇3-激酶（phosphoinositide 3-kinase, PI-3激酶）及RalGDS进行了检测。结果表明，激活Raf或RalGDS即可诱导TBP启动子活性，而激活PI-3激酶则无此效果。Raf与RalGDS介导的启动子诱导过程均需要丝裂原活化蛋白激酶激酶（mitogen-activated protein kinase kinase, MEK）的激活。此外，另一条不依赖MEK激活的Ras激活通路似乎也可诱导TBP启动子活性。对TBP启动子中负责上述调控事件的DNA序列需求进行分析后，我们确定了三个不同的区域，它们分别调控Raf、RalGDS以及Ras依赖且MEK非依赖的通路对人TBP启动子活性的调节能力。综上，本研究为TBP可在转录水平受到调控提供了新的实验证据，并明确了三条可调控这一真核核心转录因子的Ras激活通路。