Characterization of the function of gene body DNA methylation

基因体 DNA 甲基化功能的表征

• 批准号: 10394704
• 负责人: Colette Lafontaine Picard
• 金额: $ 6.98万
• 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/10394704
• 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 甲基化主要作为抑制标记。然而，DNA甲基化在基因组中也很常见。 这是一种称为基因体甲基化（GBM）的现象。GBM通常与抑制无关 相反，GBM基因倾向于适度表达，更长，功能更强 比非GBM基因更重要。GBM在整个植物和动物界也是高度保守的。的 尽管DNA甲基化具有致突变性，但GBM的广泛保守性表明， 甲基化的类型在功能上是重要的。GBM的丧失也是癌症的标志，并且可能有助于 癌细胞中的异常表型。然而，尽管有这些观察结果，GBM的功能 仍然是表观遗传学中一个基本的未决问题。提高我们对GBM功能的理解， 这不仅可以促进我们对表观遗传调控的理解，还可以提供有价值的见解， 用于癌症治疗的临床应用。 目前有三个主要的假设功能GBM。最初提出的第一个假设 基于外显子中的DNA甲基化高于内含子的观察，GBM调节 拼接另一种假设是GBM参与调节基因表达水平。最后，第三个 GBM抑制基因体内异常转录起始的假说。 直到最近，GBM不能直接干扰而不引起DNA的全基因组变化 甲基化，这限制了关于其功能的结论。然而，雅各布森实验室 现在已经开发出在拟南芥中进行靶向DNA甲基化编辑的工具。这项建议旨在 我使用一系列仔细的实验来测试GBM的潜在功能，这些新的编辑工具以及 基因组学测定。使用拟南芥作为模型系统，GBM的三种假设功能中的每一种都将 进行系统评估。最初的实验将使用全基因组测序来识别候选GBM 与野生型相比，在两个低甲基化品系中具有改变的表达、剪接或隐蔽转录的基因， 类型.这些候选GBM基因将在野生型背景下使用靶向DNA去甲基化， 通过甲基化编辑，以确认GBM的损失足以引起观察到的表型。如果这些 实验揭示了GBM的作用，潜在的机制也将被剖析。一个可能的候选人， 介导GBM依赖性调节的是MBD2，其特异性结合野生型中的GBM基因。 将进行实验以确定GBM的损失是否破坏MBD2结合，以及是否存在束缚。 在人工去甲基化GBM基因上的MBD2可以恢复正常表型。其他潜在效应物 GBM介导的调节也将被探索。综合起来，这些实验将代表 迄今为止，对GBM功能的彻底调查。 !