Role of PADI4 as a key epigenetic regulator of the p53 pathway and tumor suppression

项目总结 TP53基因是人类癌症中最常见的突变基因。虽然众所周知，P53的能力 作为转录因子是肿瘤抑制所必需的，p53下游的关键靶基因 对肿瘤抑制所需的物质仍不完全了解。此外，对p53下药的尝试已经 事实证明无效，因此需要了解肿瘤抑制途径中的下游参与者 开发新的治疗方法。墨菲实验室鉴定了三种被称为“亚型”的P53变种 P53转录功能受损最小，但罹患癌症的风险增加。这三个P53 只有少数几个靶基因的反式激活使下位基因受损，顶端靶基因受损。 为肽基-精氨酸脱氨酶4(PADI4)。PADI4是组蛋白通过瓜氨酸化修饰的调节剂， 这是将未经修饰或单甲基化的精氨酸脱亚胺为非天然氨基酸的过程 瓜氨酸。组蛋白瓜氨酸化被预测为调节基因转录，并已知在某些情况下 去凝集染色质。依赖PADI4的瓜氨酸化也参与免疫细胞的激活和 招聘。此前的报道表明，PADI4可能是一种癌基因或肿瘤抑制因子，而且 这场争议背后的原因尚不清楚。此外，还对PADI4和PADI4的作用机理进行了研究 在癌症中缺乏组蛋白瓜氨酸化。TCGA分析显示PADI4表达下调或突变 在多种人类癌症中，包括8%的黑色素瘤的突变，表明该基因是一种肿瘤 抑制者。我已经发现，在多种组织中，PADI4水平随着遗传毒性损伤的反应而增加，但 这在p53Y107H和PADI4 KO小鼠中被取消。PADI4还抑制野生型P53的增殖 类型(WT)和P53缺失的癌细胞。P53稳定剂Nutlin-3a，+/-处理细胞的RNA-SEQ分析 SI-PADI4，揭示了PADI4是转录激活p53靶基因子集所必需的。 综上所述，这些数据表明PADI4在癌症中是一种新的表观遗传调节因子，并可能发挥关键作用 在P53介导的肿瘤抑制中发挥作用。我假设PADI4通过 与P53的相互作用，并通过瓜氨酸化组蛋白和 在目标位置打开染色质。为了检验这一假设，我提出了两个目标。在第一个目标中，我将 建立PADI4在P53、WT和P53缺失黑色素瘤细胞中的直接转录靶点。我也会正式 测试PADI4增加组蛋白瓜氨酸化以引起染色质可及性改变的假设。 在第二个目的中，我将评估PADI4和PADI4靶基因在异种移植中的抑瘤能力 肿瘤生长研究。最后，虽然PADI4具有明确的转录作用，但PADI4也被认为是 增强抗原识别，但这是否有助于抑制肿瘤尚不清楚。我要测试一下这个 假设。总而言之，这项提议试图发现关于一个令人兴奋但鲜为人知的 表观遗传修饰物及其在肿瘤抑制和p53途径中的作用。

PROJECT SUMMARY TP53 is the most frequently mutated gene in human cancer. While it is well understood that the ability of p53 to act as a transcription factor is required for tumor suppression, the key target genes downstream of p53 required for tumor suppression are still incompletely understood. Moreover, attempts at drugging p53 have proven ineffective, generating a need to understand downstream players in the tumor suppressive pathway to develop novel therapies. The Murphy lab has characterized three p53 variants termed “hypomorphs” that are minimally impaired for p53 transcriptional function but have increased cancer-risk. These three p53 hypomorphs are impaired for transactivation of only a few target genes, with the top target gene impaired being peptidyl-arginine deiminase 4 (PADI4). PADI4 is a regulator of histone modification via citrullination, which is the process of deiminating unmodified or mono-methylated arginine to the non-natural amino acid citrulline. Histone citrullination is predicted to regulate gene transcription and is known in some cases to decondense chromatin. PADI4 dependent citrullination is also involved in immune cell activation and recruitment. Previous reports have suggested PADI4 may act as an oncogene or a tumor suppressor, and the reasons underlying this controversy remain unclear. Additionally, mechanistic studies on PADI4 and histone citrullination in cancer are lacking. TCGA analysis reveals that PADI4 is downregulated or mutated in multiple human cancers, including mutations in 8% of melanoma, suggesting that this gene is a tumor suppressor. I have found that PADI4 levels increase in response to genotoxic injury in multiple tissues, but this is abolished in p53Y107H and PADI4 KO mice. PADI4 also suppresses the proliferation of both p53 wild type (WT) and p53-null cancer cells. RNA-seq analysis of cells treated with the p53 stabilizer Nutlin-3a, +/- si-PADI4, reveals PADI4 to be required for transcriptional activation of a subset of p53 target genes. Collectively, these data suggest that PADI4 as a novel epigenetic regulator in cancer and may play a key role in p53-mediated tumor suppression. I hypothesize that PADI4 contributes to tumor suppression through interaction with p53, and through regulating chromatin organization by citrullinating histones and opening chromatin at targeted loci. To test this hypothesis, I propose two aims. In the first aim, I will establish the direct transcriptional targets of PADI4 in p53 WT and p53 null melanoma cells. I will also formally test the hypothesis that PADI4 increases histone citrullination to induce changes in chromatin accessibility. In the second aim, I will assess the tumor suppressive ability of PADI4 and PADI4 target genes in xenograft tumor growth studies. Finally, whereas PADI4 has a clear transcriptional role, PADI4 is also known to enhance antigen recognition, but whether this contributes to tumor suppression is unknown. I will test this hypothesis. In sum, this proposal seeks to uncover new information about an exciting but poorly understood epigenetic modifier and its role in tumor suppression and the p53 pathway.