UNS: Collaborative Research: Targeted CpG Methylation

1505793/1510652奥斯特梅尔/诺维纳人体内每个细胞都含有相同的脱氧核糖核酸序列。然而，身体中的不同类型的细胞(脑、肝、血、皮肤等)执行不同的功能。例如，在一种细胞类型中处于关闭状态的某些基因在另一种细胞类型中可能处于打开状态，从而使每个细胞具有独特的功能。基因在特定细胞中是关闭的还是开启的，部分原因是被称为CpG甲基化的DNA修饰的差异。此外，某些疾病是由CpG甲基化异常引起的。在这项研究中，将开发一种可以非常精确和可控的方式改变CpG甲基化的蛋白质。这种蛋白质将为其他研究人员提供一个工具，以研究CpG甲基化及其影响，改变细胞行为，并开发基于选择性改变单基因功能的治疗方法。一名博士后学生和一名博士后研究员将在进行这项研究的同时接受研究培训。将开发模块化的靶向胞嘧啶DNA甲基转移酶(MTase)，能够以95%的效率甲基化目标CpG位点(即5‘-CG-3’)，而不使非靶CpG位点甲基化(1%)。通过要求MTase以序列依赖的方式在所需的靶点组装成功能形式，将实现高特异性。这些MTase将使用定向进化进行优化。将开发一种用于快速评估甲基化特异性的大肠杆菌反向选择系统。为了使靶向MTase适用于哺乳动物启动子的抑制，将开发一个用户友好的平台来快速鉴定其甲基化导致真核启动子抑制的CPGS。这项工作将为鉴定真核生物中抑制启动子CpG甲基化介导的内源性启动子沉默提供一系列的分析和试剂。该流水线将针对最大的靶上基因抑制和最小的靶外基因抑制进行优化，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)研究甲基化模式的传播和表观遗传调控的机制，(3)使用靶向MTase作为工具来沉默感兴趣的基因或控制干细胞的分化，以及(4)开发靶向MTase作为选择性沉默基因(例如癌症或病毒感染)的治疗剂。该奖项由CBET部门生物技术和生化工程项目共同资助，由分子和细胞生物学部门的细胞动力学和功能项目共同资助。

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.