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

描述(申请人提供)：无法随意操纵DNA甲基化或其他表观遗传标记仍然是表观遗传学领域的最大限制之一。我们的目标是通过设计一种创新的方法来克服这一限制，在一个独特的细胞系统中对DNA甲基化进行有针对性的操作，同时也能够准确地测量这种性能。对表观遗传标记进行精确的时间和局部控制将是进一步剖析其在基因组调控中的确切功能的关键(S)。写而不是只读表观遗传标记的能力是最后一个缺乏的部分，它自信地和一般地建立功能关系，控制基因组，因此将在许多领域产生广泛影响。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甲基化的领先团队之一，我们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2000多个简化代表性亚硫酸盐测序(RRBS)和50多个全基因组亚硫酸盐测序(WGBS)数据集，为我们提供了100多种小鼠和人类细胞类型的1000亿CpG甲基化测量。因此，我们有信心地说，我们知道DNA甲基化在基因组中的哪里可以找到，以及它是如何受到基因组环境的影响的。此外，我们已经建立了许多小鼠ES细胞系，几乎涵盖了三个催化活性DNMT(1，3a和3b)及其辅助因子DNMT3l的功能丧失和/或功能获得的每一种组合。这些反过来又为这项拟议的研究提供了独特的基础，并使我们能够在控制混杂因素的同时最准确地确定效率。我们坚信，在不了解如何在基因组背景下写入这种标记的规则和原则的情况下，简单地设计工具写入基因组将永远不会提供通用的策略。

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.