UNS: Collaborative Research: Targeted CpG Methylation

人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（脑、肝、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“开启”，这使得每个细胞都具有独特的功能。基因在特定细胞中是开启还是关闭，部分是由于DNA修饰的差异，称为CpG甲基化。此外，某些疾病是由异常CpG甲基化引起的。在这项研究中，将开发一种可以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来研究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个基因功能的治疗方法。一名博士预科生和一名博士后将在进行这项研究的同时接受研究培训。模块化的靶向胞嘧啶DNA甲基转移酶（MTases）将被开发，能够以95%的效率甲基化目标CpG位点（即5'-CG-3'），同时使非目标CpG位点未甲基化（1%）。通过要求MTase以序列依赖的方式在期望的目标位点组装成功能形式，可以实现高特异性。这些mtase将使用定向进化进行优化。将开发一种用于甲基化特异性快速评估的大肠杆菌反向选择系统。为了使靶向mtase适应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识别甲基化导致真核生物启动子抑制的CpGs。这项工作将产生一系列检测和试剂，用于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。该管道将优化为最大的目标和最小的非目标基因抑制，并将扩展到全基因组应用。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传播机制，(3)使用靶向mtase作为沉默感兴趣的基因或干细胞受控分化的工具，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）的治疗药物。该奖项由CBET部门的生物技术和生化工程项目颁发，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.