Characterization of the function of gene body DNA methylation

基因体 DNA 甲基化功能的表征

• 批准号: 9910978
• 负责人: Colette Lafontaine Picard
• 金额: $ 6.49万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9910978
• 关键词: Affect; Alternative Splicing; Animals; Arabidopsis; Binding; Binding Proteins; Biological Assay; Biological Models; Cancerous; Candidate Disease Gene; Cells; ChIP-seq; Chromatin; Complementary DNA; DNA Methylation; DNA Polymerase II; Defect; Epigenetic Process; Eukaryota; Exons; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; Goals; Introns; Invertebrates; Investigation; Length; Malignant Neoplasms; Mammals; Maps; Mediating; Methylation; Nucleosomes; Pattern; Phenotype; Plants; Positioning Attribute; Property; RNA; RNA Splicing; Regulation; Regulator Genes; Repression; Role; Series; Speed; System; Techniques; Testing; Transcript; Transcription Initiation; Transcription Initiation Site; Up-Regulation; Work; base; cancer therapy; clinical application; epigenetic regulation; experimental study; gene conservation; gene function; genome-wide; global run on sequencing; improved; insight; nanopore; promoter; tool; transcriptome sequencing

Project Summary DNA methylation is an epigenetic mark found in most eukaryotes. At transposons and promoters, DNA methylation primarily acts as a repressive mark. However, DNA methylation is also commonly found over gene bodies, a phenomenon called gene body methylation (GBM). GBM is not generally associated with repression of marked genes; instead, GBM genes tend to be moderately expressed, longer, and more functionally important than non-GBM genes. GBM is also highly conserved throughout the plant and animal kingdoms. The widespread conservation of GBM in spite of the mutagenic properties of DNA methylation suggests that this type of methylation is functionally important. Loss of GBM is also a hallmark of cancer, and may contribute to the aberrant phenotypes seen in cancerous cells. Yet despite these observations, the function of GBM remains a fundamental open question in epigenetics. Improving our understanding of the function of GBM will not only advance our understanding of epigenetic regulation, but may also provide valuable insights that could be used for clinical applications in cancer treatment. There are currently three major hypothesized functions for GBM. The first hypothesis, originally proposed based on the observation that DNA methylation is higher in exons than in introns, is that GBM modulates splicing. Another hypothesis is that GBM is involved in regulating gene expression levels. Finally, a third hypothesis proposes that GBM represses aberrant transcription initiation within gene bodies. Until recently, GBM could not be directly perturbed without causing genome-wide changes in DNA methylation, which limited the conclusions that could be drawn about its function. However, the Jacobsen lab has now developed tools to perform targeted DNA methylation editing in Arabidopsis. This proposal aims to use a careful series of experiments to test potential functions of GBM using these new editing tools alongside genomics assays. Using Arabidopsis as a model system, each of the three hypothesized functions of GBM will be systematically evaluated. Initial experiments will use genome-wide sequencing to identify candidate GBM genes with altered expression, splicing, or cryptic transcription in two hypomethylated lines relative to wild- type. These candidate GBM genes will then be demethylated in a wild-type background using targeted DNA methylation editing, to confirm that loss of GBM is sufficient to cause the observed phenotype. If these experiments reveal a role for GBM, potential mechanisms will also be dissected. One likely candidate for mediating GBM-dependent regulation is MBD2, which specifically binds at GBM genes in wild-type. Experiments will be performed to determine if loss of GBM disrupts MBD2 binding, and whether tethering MBD2 at artificially demethylated GBM genes can restore a normal phenotype. Other potential effectors of GBM-mediated regulation will also be explored. Taken together, these experiments will represent the most thorough investigation of the function of GBM to date. !

项目摘要 DNA甲基化是在大多数真核生物中发现的表观遗传标记。在转座子和启动子，DNA 甲基化主要起抑制标记的作用。然而，DNA甲基化也普遍存在于基因之上 这种现象称为基因体甲基化(GBM)。GBM一般不与压抑联系在一起 标记基因；相反，GBM基因倾向于适度表达、更长且功能更强 比非GBM基因更重要。GBM在整个动植物王国中也高度保守。这个 尽管DNA甲基化具有突变特性，但GBM的广泛保守表明这一点 甲基化的类型在功能上很重要。GBM的缺失也是癌症的一个标志，并可能导致 癌细胞中出现的异常表型。然而，尽管有这些观察，GBM的功能 在表观遗传学中仍然是一个基本的悬而未决的问题。提高我们对GBM功能的认识将会 不仅增进了我们对表观遗传调控的理解，而且还可能提供有价值的见解， 可用于癌症治疗的临床应用。 目前，GBM有三个主要的假设功能。第一个假设，最初提出的 根据DNA甲基化在外显子中比内含子中更高的观察结果，GBM是否调节 拼接。另一种假设是，GBM参与了基因表达水平的调节。最后，第三个 假说认为，GBM抑制基因体内的异常转录启动。 直到最近，如果不引起全基因组的dna变化，gbm就不可能被直接干扰。 甲基化，这限制了关于其功能的结论。然而，雅各布森实验室 现在已经开发了在拟南芥中执行定向DNA甲基化编辑的工具。这项建议旨在 使用这些新的编辑工具和一系列仔细的实验来测试GBM的潜在功能 基因组学分析。以拟南芥为模型系统，GBM的三个假设功能中的每一个将 被系统地评估。最初的实验将使用全基因组测序来识别候选的GBM 与野生型相比，在两个低甲基化系中表达、剪接或隐蔽转录发生变化的基因 打字。然后，这些候选的GBM基因将在野生型背景下使用目标DNA进行去甲基化 甲基化编辑，以确认GBM的丢失足以导致观察到的表型。如果这些 实验揭示了GBM的作用，潜在的机制也将被剖析。一位可能的候选人 介导GBM依赖调节的是Mbd2，它与野生型中的GBM基因特异性结合。 将进行实验，以确定GBM的丢失是否会扰乱Mbd2的结合，以及系留是否 Mbd2人工去甲基化的GBM基因可以恢复正常的表型。其他潜在的效应器 还将探索GBM介导的监管。总而言之，这些实验将代表最大的 到目前为止对GBM的功能进行了彻底的调查。 好了！