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一致序列（p53 HH位点）结合来激活转录。尽管p53的转录抑制可能同样重要，但对于p53识别被抑制基因启动子的机制知之甚少。在迄今为止所研究的基因中，在被抑制的启动子内缺乏一致的p53结合位点，这使得研究人员提出p53间接起作用，要么通过抑制结合激活子，要么通过抑制基础机制。我们对MDR1启动子重新提出了这个问题，首先提出了一个新的假设，即p53从“激活子”到“抑制子”的“转换”可能涉及与一个非共识的DNA结合位点的相互作用；事实上，结合本身可能需要或诱导p53四聚体构象的改变，从而导致抑制结构域的暴露。基于这一假设，我们已经确定了MDR1启动子内p53半位点的新排列，它能够像共识的HH位点一样有效地与p53相互作用。重要的是，该位点（HT位点）的突变消除了p53结合和p53介导的MDR1启动子的抑制。有趣的是，p53家族成员不能通过HT位点抑制，事实上，通过一个独立的区域激活MDR1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)我们将在体外和体内分析HT-p53复合物与HH-p53复合物与转录辅助因子的相互作用；2)我们将确定p53通过HT元件参与抑制的结构域，并评估翻译后修饰对调节这种抑制的影响；3)我们将继续在MDR1启动子和其他已确定HT位点的p53抑制启动子中表征HT元件；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MDR1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.