Generation and characterization of tools for target-specific de novo DNA methylat

描述（由申请人提供）：不能随意操纵DNA甲基化或其他表观遗传标记仍然是表观遗传学领域最大的限制之一。我们的目标是通过设计一种创新的方法来克服这一限制，该方法用于在独特的细胞系统中靶向操纵DNA甲基化，并且还能够准确测量这种性能。对表观遗传标记进行精确的时间和局部控制对于进一步剖析其在基因组调控中的确切功能至关重要。书写而不仅仅是阅读表观遗传标记的能力是最后一个缺失的部分，它可以自信地建立功能关系，控制基因组，因此将对许多领域产生广泛的影响。 在过去的五年里，我的实验室一直是在全基因组范围内绘制和操纵DNA甲基化的领导小组之一，我们已经积累了可能是最大的单碱基分辨率DNA甲基化测量数据库。我们已经生成了超过2000个简化代表性亚硫酸氢盐测序（RRBS）和超过50个全基因组亚硫酸氢盐测序（WGBS）数据集，为我们提供了超过1000亿个CpG甲基化测量结果，涉及100多种小鼠和人类细胞类型。因此，我们有信心声明，我们知道在基因组中可以发现DNA甲基化的位置以及它如何受到其基因组环境的影响。此外，我们已经建立了许多小鼠ES细胞系，几乎涵盖了三种催化活性Dnmt（1、3a和3b）及其辅因子Dnmt 31的功能丧失和/或获得的每种组合。这些反过来又为这项拟议的研究提供了一个独特的基础，使我们能够最准确地确定效率，同时控制混杂因素。我们坚信，仅仅设计工具来写入基因组，而不理解如何在基因组环境中写入这种标记的规则和原则，永远不会提供一个通用的策略。

DESCRIPTION (provided by applicant): The inability to manipulate DNA methylation or other epigenetic marks at will remains one of the biggest constrains in the field of epigenetics. Our goal is to overcome this limitation by designing an innovative approach for targeted manipulation of DNA methylation in a unique cellular system that also enables accurate measurements of such performance. Having the precise temporal and localized control over epigenetic marks will be essential for further dissecting their exact function(s) in genome regulation. The ability to write rather than just read epigenetic marks is the last missing piece t confidently and generally establish functional relationships, control the genome and will therefore have a wide impact on many fields. Over the past five years my lab has been one of the leading groups to map and manipulate DNA methylation at a genome-wide scale and we have accumulated likely the largest database of DNA methylation measurements at single base resolution. We have generated well over 2000 reduced representation bisulfite sequencing (RRBS) and more than 50 whole genome bisulfite sequencing (WGBS) datasets providing us with over a 100 billion CpG methylation measurements across more than a hundred mouse and human cell types. As a result we are confident to state that we know where in the genome DNA methylation can be found and how it is influenced by its genomic environment. Moreover we have created many mouse ES cells lines covering nearly every combination of loss and/or gain of function for the three catalytically active Dnmts (1, 3a and 3b) and their co-factor Dnmt3l. These in turn provide a unique foundation for this proposed study and enables us to most accurately determine efficiencies while controlling confounding factors. We strongly believe that simply engineering tools to write onto the genome without understanding the rules and principles on how this mark can be written in a genomic context will never provide a universal strategy.