Elucidating the molecular mechanism and physiological relevance of TIRR mediated inhibition of p53

53BP1通过两个独立的机制影响基因组稳定性：(I)直接作用于DNA双链断裂 (2)增强P53作为转录因子的活性。53BP1在DSB修复中的作用 大量的文献对此进行了很好的描述。然而，对于53BP1是如何形成的，人们的理解非常有限 对P53的影响与机制和功能方面的报道不同。扰乱正常的有丝分裂导致了P53- 依赖性细胞周期停滞依赖于53BP1及其已知的相互作用因子USP28。建议的 其机制是53BP1/USP28在P53的稳定性和核定位中发挥作用。另一项研究 报道了53BP1/USP28在DNA损伤/应激反应中增强了P53的反式激活功能。 53BP1/USP28缺失会损害P53的染色质募集，但不影响P53蛋白水平。我们 最近发现的TIRR(Tudor相互作用修复调节因子)，是53BP1的直接相互作用和调节因子 在DSB修复中的作用。通过识别组蛋白H4在赖氨酸K20上的二甲基化而向DSB募集53BP1 被与53BP1的Tudor结构域结合的TIRR抑制。与p53一样，TIRR也独立地与53BP1结合 染色质和DSB。我们推测TIRR可能调节P53与53BP1的相互作用，并影响 53BP1介导的P53的激活。TIRR的缺失确实增强了53BP1和P53的相互作用，导致 P53‘S基因反式激活功能异常增强。TIRR特异性地抑制了络合物的形成 在53BP1的Tudor结构域和准备转录激活的P53的二甲基化形式之间 它的目标基因。53BP1缺失可部分挽救TIRR缺失对P53基因表达的影响 或USP28。因此，在目标1中，我们建议研究TIRR与53BP1/USP28在调控中的相互作用 P53的反式激活功能。TIRR是一种RNA结合蛋白，我们先前的结构研究揭示了两个非 TIRR中典型的RNA结合基序。我们系统地鉴定了TIRR结合的转录本，并发现 TIRR直接结合几个受P53调控的基因的转录本。结合发生在转录起始附近 这些转录本的3‘端非编码区。根据这些数据，我们假设TIRR可能会影响 转录延长和mRNA稳定性，提供了一个53BP1不依赖的机制，通过TIRR 调节P53信号轴。这一假说将在目标2中得到验证。 我们观察到TIRR基因在人类癌症中的扩增是 MDM2/4基因座的TP53突变/缺失和扩增相互排斥，表明这些 是抑制P53功能的不同机制。我们建立了一种TIRR缺陷小鼠模型来研究 认为失去TIRR将防止P53熟练的肿瘤形成的想法。我们的初步结果支持这一点 认为这些动物在p53杂合子背景下，类似于Li Fraumeni综合征(LFS)患者， 极大地减少了肿瘤的发生。在目标3中，我们将定义TIRR在癌症生物学中的相关性 生物体水平。

53BP1 influences genome stability via two independent mechanisms: (i) a direct role in DNA double-strand break (DSB) repair, (ii) enhancing the activity of p53 as a transcription factor. The role of 53BP1 in DSB repair has been well described with a large body of literature. However, there is very limited understanding of how 53BP1 impacts p53 with contrasting reports on mechanism and function. Perturbing ‘normal’ mitosis caused a p53- dependent cell cycle arrest which was dependent on 53BP1 and its known interactor USP28. The suggested mechanism was that 53BP1/USP28 played a role in the stability and nuclear localization of p53. Another study reported that 53BP1/USP28 accentuated the transactivation function of p53 in response to DNA damage/stress. Loss of 53BP1/USP28 impaired the chromatin recruitment of p53 but did not impact p53 protein levels. We recently identified TIRR (Tudor Interacting Repair Regulator), as a direct interactor and regulator of 53BP1 function in DSB repair. 53BP1 recruitment to DSBs by recognition of histone H4 dimethylated at lysine K20 is inhibited by TIRR binding to the Tudor domain of 53BP1. Like p53, TIRR also binds 53BP1 independent of chromatin and DSBs. We speculated that TIRR may regulate the interaction of p53 with 53BP1, and influence 53BP1-mediated activation of p53. Loss of TIRR indeed enhanced the interaction of 53BP1 and p53 resulting in an aberrant increase in p53’s gene-transactivation function. TIRR specifically inhibited the complex formation between the Tudor domain of 53BP1 and a dimethylated form of p53 that is poised for transcriptional activation of its target genes. The impact of TIRR loss on p53 gene expression was partially rescued by depletion of 53BP1 or USP28. Therefore in Aim 1 we propose to investigate the interplay of TIRR with 53BP1/USP28 in regulating p53 transactivation function. TIRR is a RNA binding protein and our prior structural studies revealed two non- canonical RNA binding motifs in TIRR. We systematically identified TIRR bound transcripts and discovered that TIRR directly binds transcripts of several p53-regulated genes. The binding occurs near the transcription start sites (TSS) and the 3’UTR of these transcripts. Based on this data we hypothesize that TIRR may influence transcriptional elongation and mRNA stability, providing a 53BP1-independent mechanism by which TIRR regulates the p53 signaling axis. This hypothesis will be tested in Aim 2. Exploring the clinical relevance of TIRR mediated inhibition of p53, we observed that amplification of the TIRR genomic locus in human carcinomas is mutually exclusive from TP53 mutation/deletions and amplifications of the MDM2/4 loci suggesting that these are distinct mechanisms of suppressing p53 function. We generated a TIRR-deficient mouse model to investigate the idea that loss of TIRR will prevent the formation of p53-proficient tumors. Our preliminary results support this idea as these animals in the context of p53 heterozygosity, resembling Li Fraumeni syndrome (LFS) patients, have dramatically reduced tumorigenesis. In Aim 3 we will define the relevance of TIRR in cancer biology at the organismal level.