Defining the regulation of UHRF1 and DNMT1 for maintenance of the epigenome

项目总结 在多细胞生物中，胞嘧啶5‘端的DNA甲基化有助于确保基因的正确表达。 甲基化在两条DNA链上对称地发生，维持甲基转移酶DNMT1是 负责在DNA过程中将甲基化模式从双亲链复制到子链 复制。DNMT1的维持甲基化需要染色质相关的泛素连接酶UHRF1， 它催化组蛋白H3(H3ub)的泛素化。许多问题仍然存在，关于各个领域如何 DNMT1和uhrf1的功能受到调节，这是维持DNA甲基化所必需的。这个 本提案的目的是定义uhrf1、DNMT1和H3之间的机制关系 泛素化在维持DNA甲基化中的作用。第一个目标是研究泛素化的假说 Uhrf1对组蛋白H3的作用调节DNMT1的催化活性和DNMT1维持甲基化 依赖于uhrf1泛素连接酶依赖和独立的机制。AIM 1将在体外使用 DNMT1和uhrf1中危害单个结构域的点突变的生化分析 这些蛋白质中。然后，编码这些突变酶的转基因将在癌细胞中进行测试， H3ub的印迹分析和DNA甲基化的EPIC阵列分析。这些实验将定义贡献 DNMT1和uhrf1到H3泛素化和DNA甲基化的个体功能研究 维持癌细胞的甲基化。第二个目的是检验变构激活的假说 半甲基化脱氧核糖核酸激活的S泛素连接酶在细胞中的作用及对基因组的调控 架构，与CTCF/Cohehein合作。Aim 2将使用uhrf1突变的转基因来测试heDNA 用H3ub和发夹状亚硫酸盐的蛋白质印迹分析刺激癌细胞中uhrf1‘S酶的活性 测序以确定heDNA的水平。Uhrf1功能对基因组结构的贡献 最近确定的稳定的ctcf/粘附素位点的heDNA也将使用ctcf芯片-seq和Hi-C进行定义。 这些实验将确定heDNA是否调节癌细胞中uhrf1的活性，并测试 Ctcf/粘附素位点稳定的heDNA通过uhrf1对基因组结构的贡献。监管不善 DNA甲基化是许多癌症的驱动力。因此，深入了解DNA甲基化是如何合成的 传播是了解癌症如何开始和发展的核心。这里提出的研究将 增进我们对甲基化维持机制的了解，以允许合理设计 阻止或逆转致癌DNA甲基化模式形成的治疗方法。

PROJECT SUMMARY In multicellular organisms, methylation of DNA on the 5’ carbon of cytosine helps ensure proper gene expression. Methylation occurs symmetrically on both DNA strands, and the maintenance methyltransferase DNMT1 is responsible for copying the methylation pattern from the parent strand to the daughter strand during DNA replication. Maintenance methylation by DNMT1 requires the chromatin-associated ubiquitin ligase UHRF1, which catalyzes ubiquitination of histone H3 (H3ub). Many questions remain about how the various domain functions of DNMT1 and UHRF1 are regulated and which are necessary to maintain DNA methylation. The objective of this proposal is to define the mechanistic relationship between UHRF1, DNMT1, and H3 ubiquitination in the maintenance of DNA methylation. The first aim investigates the hypothesis that ubiquitination of histone H3 by UHRF1 regulates the catalytic activity of DNMT1 and that maintenance methylation by DNMT1 depends on both UHRF1 ubiquitin ligase-dependent and independent mechanisms. Aim 1 will use in vitro biochemical assays to characterize point mutations in DNMT1 and UHRF1 that compromise individual domains of these proteins. Transgenes encoding these mutant enzymes will then be tested in cancer cells using western blot analysis of H3ub and EPIC array analysis of DNA methylation. These experiments will define the contribution of individual functions of DNMT1 and UHRF1 to H3 ubiquitination and DNA methylation in vitro and during DNA maintenance methylation in cancer cells. The second aim tests the hypothesis that allosteric activation of UHRF1’s ubiquitin ligase activity by hemimethylated DNA (heDNA) operates in cells and regulates genome architecture in cooperation with CTCF/cohesin. Aim 2 will use UHRF1 mutant transgenes to test whether heDNA stimulates UHRF1’s enzymatic activity in cancer cells using western blot analysis of H3ub and hairpin bisulfite sequencing to determine levels of heDNA. The contribution of UHRF1 function to genome architecture through recently identified stable heDNA at CTCF/cohesin sites will also be defined using CTCF ChIP-seq and Hi-C. These experiments will determine if heDNA regulates the activity of UHRF1 in cancer cells and test the contribution of stable heDNA at CTCF/cohesin sites to genome architecture through UHRF1. Misregulation of DNA methylation is a driving force in many cancers. Thus, insight into how DNA methylation is synthesized and propagated is central to understanding how cancer begins and progresses. The studies proposed here will advance our knowledge of the mechanisms involved in methylation maintenance to allow the rational design of therapies that halt or reverse the formation of oncogenic DNA methylation patterns.