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

定义 UHRF1 和 DNMT1 对表观基因组维护的调控

基本信息

批准号：
10604368
负责人：
Joel Hrit
金额：
$ 7.18万
依托单位：
VAN ANDEL RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10604368
关键词：
3-Dimensional Aberrant DNA Methylation Antibodies Architecture Binding Binding Sites Biochemical Biochemistry Biological Assay Biology CCCTC-binding factor Carbon Catalysis Categories Cell division Cell physiology Cells ChIP-seq Chemicals Chromatin Complex CpG dinucleotide Cytosine DNA DNA Maintenance DNA Methylation DNA Modification Methylases DNA Modification Process DNA analysis DNA biosynthesis DNMT3B gene DNMT3a Data Daughter Deposition Development Disease Progression Ensure Enzymes Epigenetic Process Failure Gene Expression Gene Expression Regulation Genetic Transcription Genome Genomic Imprinting Goals Hi-C Histone H3 Histones Human In Vitro Individual Knowledge Maintenance Malignant Neoplasms Methylation Methyltransferase Modeling Modification Molecular Molecular Abnormality Mutation N-terminal Nucleosomes Oncogenic Organism Parents Point Mutation Process Proteins Reagent Recombinants Regulation Role Site Specificity Structure Tail Tertiary Protein Structure Testing Transcriptional Silencer Elements Transgenes Ubiquitin Ubiquitination Western Blotting Work X Inactivation biochemical tools bisulfite sequencing cancer cell cohesin daughter strand defined contribution density driving force epigenome experimental study falls genomic locus insight loss of function mutation methyl group methylation pattern mutant novel programs rational design recruit targeted treatment therapy design therapy resistant tool tumorigenesis ubiquitin ligase ubiquitin-protein ligase

项目摘要

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.

项目摘要在多细胞生物中，胞嘧啶5'碳上DNA的甲基化有助于确保适当的基因表达。甲基化在两个DNA链上对称发生，维持甲基转移酶DNMT1 IS 负责在DNA期间将甲基化模式从父链复制到女儿链复制。 DNMT1维持甲基化需要染色质相关的泛素连接酶UHRF1，这催化了Hisstone H3（H3UB）的泛素化。关于各个领域的方式仍然存在许多问题 DNMT1和UHRF1的功能受到调节，并且对于维持DNA甲基化是必要的。这该建议的目的是定义UHRF1，DNMT1和H3之间的机械关系维持DNA甲基化的泛素化。第一个目的调查了泛素化的假设 UHRF1的组蛋白H3调节DNMT1的催化活性和DNMT1的维持甲基化取决于UHRF1泛素连接酶依赖性和独立机制。 AIM 1将在体外使用生化分析以表征DNMT1和UHRF1中损害各个领域的点突变这些蛋白质。然后，使用西方在癌细胞中测试编码这些突变酶的转基因 H3UB的印迹分析和DNA甲基化的史诗阵列分析。这些实验将定义贡献 DNMT1和UHRF1至H3泛素化和DNA甲基化的单个功能在体外和DNA期间癌细胞中的维持甲基化。第二个目的检验了以下假设半甲基化DNA（HEDNA）在细胞中起作用并调节基因组的UHRF1的泛素连接酶活性与CTCF/粘蛋白合作的建筑。 AIM 2将使用UHRF1突变体翻译来测试HEDNA是否使用H3ub和Hairpin Bisulfite的Western印迹分析刺激UHRF1在癌细胞中的酶活性测序以确定HEDNA的水平。 UHRF1功能对基因组架构的贡献最近还将使用CTCF芯片seq和hi-c来定义最近确定的在CTCF/粘着蛋白位点的稳定HEDNA。这些实验将确定HEDNA是否调节癌细胞中UHRF1的活性，并测试通过UHRF1，稳定的HEDNA在CTCF/粘着蛋白位点对基因组结构的贡献。误导 DNA甲基化是许多癌症的驱动力。这是关于如何合成DNA甲基化的洞察力传播对于了解癌症的开始和进展是至关重要的。这里提出的研究将促进我们对甲基化维持涉及的机制的了解，以允许合理设计停止或逆转致癌DNA甲基化模式的疗法。