Suppression of p53 response by blocking p53-Mediator binding with a stapled peptide. Suppression of p53 response by blocking p53-Mediator binding with a stapled peptide

DNA-binding transcription factors (TFs) have been challenging to target with molecular probes. Many TFs function in part through interaction with Mediator; we sought to block p53 function by disrupting the p53-Mediator interaction. Through rational design and activity-based screening, we characterized a stapled peptide, with functional mimics of both p53 activation domains, that selectively disrupted p53- and Mediator-dependent transcription in vitro. This “bivalent peptide” also suppressed p53 transcriptional response in human cancer cells. Our strategy circumvents the TF and instead targets the TF-Mediator interface, with desired transcriptional outcomes. Different TFs target Mediator through different subunits, suggesting this strategy could be generalizable. Overall design: Examination of polII (pol II Ser5 3E8) TSS occupancy by ChIPseq with and without the inhibitory peptide in the presense of the p53 activator Nutlin3a.

DNA结合转录因子（DNA-binding transcription factors, TFs）一直是分子探针靶向调控的难点。诸多转录因子的部分功能依赖于与中介体（Mediator）的相互作用；我们尝试通过阻断p53与中介体的相互作用，以抑制p53的生物学功能。通过理性设计与基于活性的筛选，我们获得并表征了一种钉合肽（stapled peptide），该肽携带有p53两个激活结构域的功能模拟序列，可在体外选择性阻断依赖于p53与中介体的转录过程。这款“双价肽”还可在人类癌细胞中抑制p53的转录应答。我们的策略并未直接靶向转录因子，而是靶向转录因子-中介体的相互作用界面，可实现预期的转录调控效果。由于不同转录因子通过不同亚基靶向中介体，这一策略具备通用可行性。总体实验设计：在p53激活剂Nutlin3a存在的条件下，通过染色质免疫沉淀测序（ChIP-seq）检测添加与未添加抑制性钉合肽时，RNA聚合酶II（pol II，Ser5位点3E8抗体）在转录起始位点（TSS）的占据情况。