Novel mode of p53 mediated repression the mdri paradigm

p53通过作为四聚体与靶基因启动子内的共有序列（p53 HH位点）结合来激活转录。 虽然p53的转录抑制可能同样重要，但对p53识别受抑制基因启动子的机制知之甚少。 在迄今为止研究的基因中，由于受抑制的启动子内缺乏一致的p53结合位点，研究人员提出p53间接起作用，要么通过抑制结合的激活剂，要么通过抑制基础机制。 我们已经重新解决了这个问题，相对于MDR 1启动子，开始与新的假设，即“开关”的p53从“激活剂”到“阻遏物”可能涉及与非共识DNA结合位点的相互作用，事实上，结合本身可能需要或诱导改变构象的p53四聚体，导致阻遏结构域的解蔽。 基于这一假设，我们已经确定了一种新的排列p53半位点内的MDR 1启动子，能够有效地与p53的共识HH网站。 重要的是，该位点（HT位点）的突变消除了p53结合和p53介导的MDR 1启动子抑制。 有趣的是，p53家族成员无法通过HT位点进行抑制，事实上，通过一个独立区域激活了MDR 1转录。我们建议继续研究p53通过HT位点介导的抑制，目的如下：1）我们将分析HT-p53复合物与HH-p53复合物与转录辅因子的相互作用，包括体外和体内：2）我们将确定p53通过HT元件参与阻遏的结构域，并评估后处理的效果。3）我们将继续表征HT元件，包括在MDR 1启动子和其他p53抑制的启动子中，其中我们已经鉴定了HT位点; 4）我们将通过分析p73/p53嵌合蛋白来进一步观察p53及其家族成员对MDR 1转录的不同影响。

p53 activates transcription by binding as a tetramer to a consensus sequence (the p53 HH site) within the promoter of target genes. Although transcriptional repression by p53 may be equally important, less is known about the mechanism by which p53 recognizes promoters of repressed genes. In the genes studied to date, the lack of a consensus p53 binding site within the repressed promoter has led investigators to propose that p53 works indirectly, either by inhibiting a bound activator or by repressing the basal machinery. We have re-addressed this question with respect to the MDR1 promoter, beginning with the novel hypothesis that the "switch" of p53 from an "activator" to a "repressor" may involve interaction with a non-consensus DNA binding site; in fact, binding itself may require or induce an altered conformation of the p53 tetramer, resulting in the unmasking of repressor domains. Based on this hypothesis, we have identified a novel arrangement of p53 half-sites within the MDR1 promoter which are capable of interacting with p53 as efficiently as the consensus HH site. Importantly, mutation of this site (the HT site) abrogates both p53 binding and p53- mediated repression of the MDR1 promoter. Interestingly, the p53 family members are not able to repress through the HT site and, in fact activate MDR1 transcription though an independent region. We propose to continue to investigate p53-mediated repression through the HT site in the following Aims: 1) We will analyze the interaction of the HT-p53 complex versus the HH-p53 complex with transcriptional co-factors, both in vitro and in vivo: 2) We will determine the domains of p53 involved in repression through the HT element and evaluate the effect of post-translational modifications on modulating this repression; 3) We will continue to characterize the HT element, both in the MDR1 promoter and in other p53-repressed promoters in which we have identified HT sites; and 4) We will pursue our novel observation of a diverse effect of p53 and its family members on MDR1 transcription by analyzing p73/p53 chimeric proteins.