Characterization of the function of gene body DNA methylation

基因体 DNA 甲基化功能的表征

基本信息

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
项目类别：
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

项目摘要

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。在转座和启动子，DNA 甲基化主要充当抑制标记。然而，DNA甲基化也通常在基因上发现身体，一种称为基因体甲基化（GBM）的现象。 GBM通常与抑制无关明显的基因；相反，GBM基因倾向于中度表达，更长和更有功能比非GBM基因重要。 GBM在整个动植物王国中也高度保守。这尽管DNA甲基化的诱变特性，但GBM的广泛保护表明这一点甲基化的类型在功能上很重要。 GBM的损失也是癌症的标志，可能有助于在癌细胞中看到的异常表型。尽管有这些观察，但GBM的功能在表观遗传学中仍然是一个基本的公开问题。提高我们对GBM功能的理解将不仅可以提高我们对表观遗传调节的理解，而且还可以提供有价值的见解用于癌症治疗中的临床应用。 GBM目前有三个主要的假设功能。第一个假设，最初提出基于外显子中的DNA甲基化高于内含子中的DNA甲基化的观察结果，GBM调节了剪接。另一个假设是GBM参与调节基因表达水平。最后，第三假设提出，GBM抑制基因体内异常转录起始。直到最近，GBM在不引起全基因组DNA变化的情况下才能直接受到干扰甲基化，限制了可以得出有关其功能的结论。但是，雅各布森实验室现在已经开发了在拟南芥中执行靶向DNA甲基化编辑的工具。该建议旨在使用一系列仔细的实验来测试GBM的潜在功能，并在基因组学测定。使用拟南芥作为模型系统，GBM的三个假设功能中的每一个都将进行系统评估。最初的实验将使用全基因组测序来识别候选GBM 相对于野生 - 类型。然后，这些候选GBM基因将在野生型背景中使用靶向DNA进行脱甲基甲基化编辑以确认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，还将解剖潜在的机制。一个可能的候选人介导GBM依赖性调节是MBD2，它在野生型中特异性结合了GBM基因。将进行实验以确定GBM的损失是否破坏MBD2结合，以及是否束缚人为地脱甲基的GBM基因的MBD2可以恢复正常的表型。其他潜在效应子还将探讨GBM介导的法规。综上所述，这些实验将最多代表迄今为止对GBM的功能进行了详尽的研究。呢