Crosstalk of DNMT1 and Sirt1 in Autosomal Dominant Polycystic Kidney Disease

项目摘要 常染色体显性遗传性多囊肾病（ADPKD）囊肿形成的机制仍然存在 对ADPKD的认识不完全，目前尚无有效的临床治疗方法。我的长期 我的职业目标是建立一个独立的翻译研究实验室，在那里我将阐明 囊肿形成和生长的机制，并发现ADPKD的新治疗策略。这 NIDDK指导研究科学家发展（K 01）申请提出了一项多学科的5- 一年的培训计划，为候选人夏舟博士提供经验和资源 这是开启成功职业生涯的必要条件。培训计划，与主要导师博士密切合作。 李晓刚和共同导师James Calvet博士将加强我以前在表观遗传学方面的专业知识 和ADPKD研究通过推进我的知识和技术相关的DNA研究 甲基化和组蛋白乙酰化。在堪萨斯大学非常刺激的科学环境 医疗中心将不仅为我提供必要的专业知识和设施， 完成这个项目，但也将准备我顺利过渡到一个独立的教师 位置这项研究的目的是了解一个关键的表观遗传的功能作用， ADPKD中的调节因子DNA甲基转移酶1（DNMT1）。我们的初步研究表明， DNMT 1在Pkd 1突变的肾上皮细胞和组织中上调， DNMT1抑制剂，5-氮杂胞苷和肼苯哒嗪，延迟Pkd1条件性敲除中的囊肿生长 小鼠我们先前的研究表明SIRT1的上调通过以下途径促进囊肿的发育： 在ADPKD动物模型中调节囊性肾上皮细胞增殖和死亡表明， 通过使用烟酰胺（维生素B3）治疗ADPKD的有前途的治疗策略。SIRT1具有 已发现使DNMT 1脱乙酰并增加其甲基转移酶活性。因此，我们假设 DNMT1的上调通过STAT3的激活来调节囊性肾上皮细胞的增殖 并通过p53信号调节细胞凋亡，靶向DNMT1在体内延迟肾囊肿生长， ADPKD的小鼠模型，以及DNMT 1与SIRT 1协同调节囊肿生长 在ADPKD中，靶向SIRT1和DNMT1将进一步延迟ADPKD中的囊肿生长。我们将 用三个具体目标来检验这个假设。在具体目标1中，我们将1）研究DNMT 1是否 通过SHP-1介导的磷酸化调节囊性肾上皮细胞增殖， STAT3的激活; 2）研究DNMT 1是否通过p53调节细胞凋亡 依赖路径在具体目标2中，我们将1）测试DNMT 1敲除是否会延迟肾囊肿 在Pkd 1敲除小鼠中的生长;和2）测试用5-氮杂胞苷或 肼苯哒嗪延迟Pkd1基因敲除小鼠模型中肾囊肿的生长。在具体目标3中，我们将1） 研究SIRT1是否调节囊性肾上皮细胞中DNMT1的活性和稳定性; 以及2）研究SIRT1是否调节SHP-1的甲基化状态，以及DNMT1是否调节SHP-1的甲基化状态。 调节SHP-1的组蛋白乙酰化;和3）测试DNMT 1和SIRT 1两者的抑制是否 协同延迟Pkd1基因敲除小鼠的囊肿生长。这是第一个研究，以确定功能 DNMT1和DNMT1介导的信号通路在ADPKD囊肿发育中的作用， 这不仅加深了我们对囊肿发育的理解，而且为使用DNMT 1提供了理论基础。 抑制剂作为ADPKD的疗法。此外，本研究还将揭示DNMT 1- 介导的甲基化和SIRT1介导的乙酰化调节囊肿的发展，并将有助于 促进靶向DNMT 1和SIRT 1的新型治疗方法的开发。

Project Summary The mechanisms of cyst formation in autosomal dominant polycystic kidney disease (ADPKD) remain incompletely understood and the effective clinical therapies for ADPKD are not available. My long-term career goal is to establish an independent translational research laboratory, where I will elucidate the mechanisms of cyst formation and growth, and discover novel therapeutic strategies for ADPKD. This NIDDK Mentored Research Scientist Development (K01) application proposes a multidisciplinary 5- year training program to provide the candidate Dr. Xia Zhou with the experience and resources necessary to launch a successful career. The training plan, developed closely with primary mentor Dr. Xiaogang Li and co-Mentor Dr. James Calvet will strengthen my previous expertise in the epigenetics and ADPKD research by advancing my knowledge and technologies related to the study of DNA methylation and histone acetylation. The very stimulating scientific environment at University of Kansas Medical Center will not only provide me with the expertise and facilities necessary for successful completion of this project, but will also prepare me to transition smoothly into an independent faculty position. The objective of the proposed study is to understand the functional roles of a key epigenetic regulator, DNA methyltransferase 1 (DNMT1), in ADPKD. Our preliminary studies indicated that DNMT1 was upregulated in Pkd1 mutant renal epithelial cells and tissues, and that treatment with DNMT1 inhibitors, 5-azacytidine and hydralazine, delayed cyst growth in Pkd1 conditional knockout mice. Our previous study showing that upregulation of SIRT1 contributes to cyst development through regulating cystic renal epithelial cell proliferation and death in ADPKD animal models suggests a promising therapeutic strategy for ADPKD treatment by using nicotinamide (vitamin B3). SIRT1 has been found to deacetylate DNMT1 and increase its methyltransferase activity. Thus, we hypothesize that upregulation of DNMT1 regulates cystic renal epithelial cell proliferation through STAT3 activation and regulates apoptosis through p53 signaling, and targeting DNMT1 delays renal cyst growth in vivo in orthologous murine models of ADPKD, and that DNMT1 synergizes with SIRT1 to regulate cyst growth in ADPKD, and targeting both SIRT1 and DNMT1 would delay cyst growth further in ADPKD. We will test this hypothesis with three specific aims. In specific aim 1, we will 1) investigate whether DNMT1 regulates cystic renal epithelial cell proliferation through SHP-1 mediated phosphorylation and activation of STAT3; and 2) investigate whether DNMT1 regulates apoptosis through the p53 dependent pathway. In specific aim 2, we will 1) test whether knockout of DNMT1 delays renal cyst growth in Pkd1 knockout mice; and 2) test whether inhibition of DNMT1 with 5-azacytidine or hydralazine delays renal cyst growth in Pkd1 knockout mouse models. In specific aim 3 we will 1) investigate whether SIRT1 regulates the activity and stability of DNMT1 in cystic renal epithelial cells; and 2) investigate whether SIRT1 regulates the methylation status of SHP-1, and whether DNMT1 regulates histone acetylation of SHP-1; and 3) test whether inhibition of both DNMT1 and SIRT1 synergistically delays cyst growth in Pkd1 knockout mice. This is the first study to define the functional roles of DNMT1 and DNMT1-mediated signaling pathways in cyst development in ADPKD, which will not only further our understanding of cyst development but also provide a rationale for using DNMT1 inhibitors as a therapy for ADPKD. In addition, this study will reveal the relationship between DNMT1- mediated methylation and SIRT1-mediated acetylation in regulating cyst development, and will help promote the development of novel therapeutic approaches targeting both DNMT1 and SIRT1.