UNS: Collaborative Research: Targeted CpG Methylation

1505793/1510652 Ostermeier/Novina体内的每个细胞都含有相同的DNA序列。然而，身体中不同类型的细胞（大脑，肝脏，血液，皮肤等）执行不同的功能。例如，在一种细胞类型中“关闭”的某些基因可能在另一种细胞类型中“打开”，这使得每个细胞都具有独特的功能。一个基因在特定细胞中是关闭还是打开，部分原因是DNA修饰的差异，称为CpG甲基化。 此外，某些疾病是由异常的CpG甲基化引起的。 在这项研究中，将开发一种蛋白质，可以以非常精确和可控的方式改变CpG甲基化。 这种蛋白质将为其他研究人员提供一种研究CpG甲基化及其影响的工具，以改变细胞行为，并开发基于选择性改变单个基因功能的疗法。一名博士前学生和一名博士后研究员将在进行这项研究时接受研究培训。 将开发模块化的靶向胞嘧啶DNA甲基转移酶（MT酶），其能够以95%的效率甲基化靶CpG位点（即5 '-CG-3'），同时使非靶CpG位点未甲基化（1%）。高特异性将通过要求MTase以序列依赖性方式在所需靶位点组装成功能形式来实现。这些MTase将使用定向进化进行优化。大肠大肠杆菌反向选择系统将被开发用于快速评估甲基化特异性。为了使靶向的MT酶适用于哺乳动物启动子的阻遏，将开发用于快速鉴定CpG的用户友好的平台，所述CpG的甲基化导致真核启动子的阻遏。这项工作将导致一个管道的测定和试剂，用于确定抑制性启动子CpG甲基化介导的内源性启动子在真核生物中的沉默。该管道将针对最大的靶向和最小的脱靶基因抑制进行优化，并将可扩展用于全基因组应用。我们的研究活动将使研究人员能够：（1）研究甲基化对转录的影响，（2）研究甲基化模式和表观遗传调控的扩散机制，（3）使用靶向的MT酶作为沉默感兴趣的基因或控制干细胞分化的工具，和（4）开发靶向MT酶作为选择性基因沉默的治疗剂（例如癌症或病毒感染）。CBET部门生物技术和生物化学工程项目的这一奖项是共同的，由分子和细胞生物学部的细胞动力学和功能计划资助。

1505793/1510652 Ostermeier/NovinaEvery cell in the body contains the same DNA sequence. However, the different types of cells in the body (brain, liver, blood, skin, etc.) perform different functions. For example, certain genes that are 'off' in one cell type might be 'on' in another, which enables each cell's unique function. Whether a gene is off or on in a particular cell is due in part to differences in a modification of DNA called CpG methylation. Additionally, certain diseases are caused by abnormal CpG methylation. In this research, a protein will be developed that can alter CpG methylation in a very precise and controlled fashion. This protein will provide other researchers with a tool to study CpG methylation and its effects, to alter cell behavior, and to develop therapies based on the selective altering of a single gene function. A pre-doctoral student and a postdoctoral fellow will be trained in research while performing this research. Modular, targeted cytosine DNA methyltransferases (MTases) will be developed that are capable of methylating a target CpG site (i.e. 5'-CG-3') at 95% efficiency while leaving non-target CpG sites unmethylated ( 1%). High specificity will be achieved by requiring that the MTase assembles into a functional form at the desired target site in a sequence-dependent manner. These MTases will be optimized using directed evolution. An E. coli reverse selection system will be developed for the rapid assessment of methylation specificity. To adapt the targeted MTases for the repression of mammalian promoters, a user-friendly platform for rapidly identifying CpGs whose methylation leads to repression of eukaryotic promoters will be developed. This work will result in a pipeline of assays and reagents for identifying repressive promoter CpG methylation mediated silencing of endogenous promoters in eukaryotes. The pipeline will be optimized for maximal on-target and minimal off-target gene repression and will be scalable for genome-wide applications. Our research activities will empower researchers to: (1) study methylation's effect on transcription, (2) investigate the mechanisms of spreading of methylation patterns and epigenetic regulation, (3) use targeted MTases as a tool for silencing genes of interest or controlled differentiation of stem cells, and (4) develop targeted MTases as therapeutic agents for the selective silencing of genes (e.g. cancers or viral infections).This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the Cellular Dynamics and Function Program of the Division of Molecular and Cellular Biology.