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

53 BP 1通过两种独立的机制影响基因组稳定性：（i）直接参与DNA双链断裂 (DSB)修复，（ii）增强p53作为转录因子的活性。53 BP 1在DSB修复中的作用 被大量的文学作品很好地描述了。然而，对53 BP 1如何产生的理解非常有限。 影响p53与机制和功能的对比报告。干扰“正常”有丝分裂导致p53- 依赖于53 BP 1及其已知的相互作用物USP 28的依赖性细胞周期停滞。建议的 其机制可能是53 BP 1/USP 28在p53的稳定性和核定位中起作用。另一项研究 报道53 BP 1/USP 28增强了p53对DNA损伤/应激的反式激活功能。 53 BP 1/USP 28的丢失损害了p53的染色质募集，但不影响p53蛋白水平。我们 最近鉴定出TIRR（Tudor Interacting Repair Regulator）作为53 BP 1的直接相互作用物和调节物 在DSB修复中发挥作用。通过识别在赖氨酸K20处二甲基化的组蛋白H4将53 BP 1募集至DSB， 通过与53 BP 1的Tudor结构域结合的TIRR抑制。与p53一样，TIRR也与53 BP 1结合，不依赖于 染色质和DSB。我们推测TIRR可能调节p53与53 BP 1的相互作用，并影响p53与53 BP 1的相互作用。 53 BP 1介导的p53活化。TIRR的缺失确实增强了53 BP 1和p53的相互作用， p53基因反式激活功能的异常增加。TIRR特异性抑制复合物的形成 53 BP 1的Tudor结构域和准备转录激活的p53的二甲基化形式之间的关系 它的目标基因。TIRR缺失对p53基因表达的影响可通过53 BP 1的缺失部分挽救。 或USP 28。因此，在目的1中，我们提出研究TIRR与53 BP 1/USP 28在调节细胞凋亡中的相互作用。 p53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中的典型RNA结合基序。我们系统地鉴定了TIRR结合的转录本，发现 TIRR直接结合几个p53调节基因的转录本。结合发生在转录起始点附近 这些转录物的3 'UTR和TSS位点。基于这些数据，我们假设TIRR可能会影响 转录延长和mRNA稳定性，提供了一种53 BP 1非依赖性机制，TIRR 调节p53信号传导轴。目标2将检验这一假设。探索TIRR的临床相关性 通过p53介导的抑制，我们观察到在人类癌中TIRR基因组位点的扩增， 与TP 53突变/缺失和MDM 2/4基因座扩增相互排斥，这表明这些突变/缺失和扩增可能与其他突变/缺失相关。 是抑制p53功能的不同机制。我们建立了一个TIR缺陷的小鼠模型来研究 TIRR的缺失将阻止p53活性肿瘤的形成。我们的初步结果支持这一点 这种想法是因为这些动物在p53杂合性的背景下，类似于Li Fraumeni综合征（LFS）患者， 大大减少了肿瘤的发生。在目标3中，我们将定义TIRR在癌症生物学中的相关性， 有机体水平。

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.