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. !

项目概要 DNA 甲基化是大多数真核生物中发现的表观遗传标记。在转座子和启动子处，DNA 甲基化主要充当抑制标记。然而，DNA 甲基化也常见于基因体，这种现象称为基因体甲基化（GBM）。 GBM 通常与压抑无关标记基因；相反，GBM 基因往往表达适度、更长且功能更丰富比非 GBM 基因重要。 GBM 在整个植物界和动物界中也高度保守。这尽管 DNA 甲基化具有诱变特性，但 GBM 的广泛保守性表明，甲基化的类型在功能上很重要。 GBM 缺失也是癌症的一个标志，并可能导致在癌细胞中看到的异常表型。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悬而未决的问题。提高我们对 GBM 功能的理解将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 3 个主要的假设功能。第一个假设，最初提出基于外显子中 DNA 甲基化高于内含子的观察，GBM 调节拼接。另一个假设是 GBM 参与调节基因表达水平。最后还有第三个假设提出 GBM 抑制基因体内异常的转录起始。直到最近，GBM 还无法在不引起 DNA 基因组范围变化的情况下直接受到干扰甲基化，这限制了对其功能可以得出的结论。然而，雅各布森实验室目前已开发出在拟南芥中进行靶向 DNA 甲基化编辑的工具。该提案旨在使用这些新的编辑工具进行一系列仔细的实验来测试 GBM 的潜在功能基因组学分析。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。初步实验将使用全基因组测序来识别候选 GBM 相对于野生型，两个低甲基化品系中表达、剪接或隐秘转录发生改变的基因类型。然后，这些候选 GBM 基因将在野生型背景下使用靶向 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 依赖性调节的是 MBD2，它在野生型中特异性结合 GBM 基因。将进行实验以确定 GBM 的丢失是否会破坏 MBD2 结合，以及束缚是否会破坏 MBD2在人工去甲基化的GBM基因上可以恢复正常的表型。其他潜在效应器还将探讨 GBM 介导的调节。总的来说，这些实验将代表最迄今为止对 GBM 功能的彻底研究。！