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

项目摘要 在多细胞生物中，胞嘧啶5'碳上的DNA甲基化有助于确保适当的基因表达。 甲基化对称地发生在两条DNA链上，并且维持甲基转移酶DNMT 1是 负责在DNA合成过程中将甲基化模式从母链复制到子链， 复制的DNMT 1的甲基化需要染色质相关的泛素连接酶UHRF 1， 其催化组蛋白H3（H3 ub）的泛素化。许多问题仍然存在， DNMT 1和UHRF 1的功能受到调节，并且是维持DNA甲基化所必需的。的 本提案的目的是确定UHRF 1、DNMT 1和H3之间的机制关系 泛素化在维持DNA甲基化中的作用。第一个目的是研究泛素化的假说， UHRF 1对组蛋白H3的甲基化调节DNMT 1的催化活性，并维持DNMT 1的甲基化 依赖于UHRF 1泛素连接酶依赖性和非依赖性机制。Aim 1将在体外使用 表征DNMT 1和UHRF 1中损害单个结构域的点突变的生化测定 这些蛋白质。编码这些突变酶的转基因将在癌细胞中进行检测， H3 ub的印迹分析和DNA甲基化的EPIC阵列分析。这些实验将定义 DNMT 1和UHRF 1到H3泛素化和DNA甲基化在体外和DNA合成过程中的单独功能 维持癌细胞的甲基化。第二个目的是测试的假设，变构激活的 UHRF 1通过半甲基化DNA（heDNA）的泛素连接酶活性在细胞中起作用并调节基因组 与CTCF/cohesin合作构建。目的2将使用UHRF 1突变转基因来测试heDNA是否 使用H3 ub和发夹亚硫酸氢盐的蛋白质印迹分析刺激癌细胞中UHRF 1的酶活性 测序以确定heDNA的水平。UHRF 1功能对基因组结构的贡献 还将使用CTCF ChIP-seq和Hi-C来定义最近鉴定的CTCF/粘附素位点处的稳定heDNA。 这些实验将确定heDNA是否调节癌细胞中UHRF 1的活性，并测试其对肿瘤细胞的影响。 CTCF/粘附素位点上稳定的heDNA通过UHRF 1对基因组结构的贡献。的误调节 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， 传播是理解癌症如何开始和发展的核心。本文提出的研究将 推进我们对甲基化维持机制的认识，以合理设计 阻止或逆转致癌DNA甲基化模式形成的疗法。

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.