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

项目摘要 TP 53是人类癌症中最常见的突变基因。虽然很好地理解了p53的能力， 作为肿瘤抑制所需的转录因子，p53下游的关键靶基因 对肿瘤抑制所需的机制仍不完全了解。此外，对p53进行药物治疗的尝试 被证明是无效的，需要了解肿瘤抑制途径中的下游参与者 来开发新的疗法。墨菲实验室已经鉴定了三种称为“亚型”的p53变体， 对p53转录功能的损害最小，但具有增加的癌症风险。这三个p53 亚型仅在少数靶基因的反式激活方面受损，最高的靶基因受损 为肽基精氨酸脱亚胺酶4（PADI 4）。PADI 4是通过瓜氨酸修饰组蛋白的调节剂， 该方法是将未修饰的或单甲基化的精氨酸脱亚氨基化为非天然氨基酸 瓜氨酸组蛋白瓜氨酸被预测调节基因转录，并且已知在某些情况下， 去致密染色质。PADI 4依赖性瓜氨酸还参与免疫细胞活化， 招聘先前的报道表明PADI 4可能作为癌基因或肿瘤抑制基因， 这一争议背后的原因尚不清楚。此外，对PADI 4和 组蛋白瓜氨酸缺乏。TCGA分析显示PADI 4下调或突变 在多种人类癌症中，包括8%的黑色素瘤中的突变，表明该基因是一种肿瘤。 抑制器。我发现，在多种组织中，PADI 4水平在对遗传毒性损伤的反应中增加， 这在p53 Y107 H和PADI 4 KO小鼠中被消除。PADI 4还抑制p53野生型和p53野生型细胞的增殖。 型（WT）和p53缺失癌细胞。用p53稳定剂Nutlin-3a +/-处理的细胞的RNA-seq分析 si-PADI 4揭示了PADI 4是p53靶基因亚组转录激活所需的。 总的来说，这些数据表明，PADI 4作为一种新的表观遗传调节因子在癌症中可能发挥关键作用， p53介导的肿瘤抑制。我假设PADI 4通过以下途径促进肿瘤抑制： 与p53相互作用，并通过瓜氨酸化组蛋白调节染色质组织， 在靶位点打开染色质。为了验证这个假设，我提出了两个目标。在第一个目标中，我将 在p53 WT和p53缺失黑素瘤细胞中建立PADI 4的直接转录靶标。我也会正式 检验PADI 4增加组蛋白瓜氨酸以诱导染色质可接近性变化的假设。 在第二个目标中，我将评估PADI 4和PADI 4靶基因在异种移植物中的肿瘤抑制能力 肿瘤生长研究。最后，尽管PADI 4具有明确的转录作用，但也已知PADI 4 增强抗原识别，但这是否有助于肿瘤抑制尚不清楚。我来测试一下 假说.总之，这项提案旨在揭示一个令人兴奋但知之甚少的新信息。 表观遗传修饰物及其在肿瘤抑制和p53通路中的作用。

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.