Investigating the role of long non-coding RNAs in regulation of DNA methylation

项目摘要 异常的DNA甲基化模式是癌症的早期标志。在健康的细胞中，有“作家”， 建立这些甲基化标记和“橡皮擦”，在特定的细胞类型和不同的点上去除它们 在细胞分化过程中。DNA甲基化的适当调节对正常细胞功能至关重要。一个重要 DNA甲基转移酶（DNMT）是一种重要的酶。DNMT负责建立DNA 甲基化模式以及在连续的DNA复制过程中保持它们。DNMT通常 突变的癌症和DNMT如何促进基因特异性DNA甲基化的精确机制， 健康的细胞还没有被很好地理解。最近的证据表明，RNA在 调节表观遗传标记例如，几种lncRNA已显示与DNMT相互作用，主要是 维持甲基化酶DNMT 1。在这个扩展建议中，我的目的是研究DNMT 1的调节 的RNA。我将首先阐明RNA如何与DNMT 1相互作用的分子基础， DNMT 1结合所需的核心结构特征。我将根据我收集到的初步数据采取后续行动 在我的K 00基金期间，包括发现DNMT 1对富含GU的RNA的高亲和力。另外，我有 发现DNMT 1以高亲和力结合其自身的mRNA，特别是3' UTR，有趣的是， 非常富有。我将通过对富含DNMT 1/GU的细胞进行冷冻电镜， RNA复合物以及其他生化测定，以更好地了解DNMT 1的结合偏好 for various各种RNA RNA RNA RNA.总之，这一提议将有助于阐明DNA甲基化在癌症中是如何被错误调节的， 将另外提供有关RNA如何帮助调节这一重要过程的信息。占据 了解RNA如何与DNMT 1相互作用的分子基础可能会提供结构靶点， 组织特异性癌症治疗的发展。

Project Summary Aberrant DNA methylation patterns are early hallmarks of cancer. In healthy cells, there are “writers” that establish these methylation marks and “erasers” that remove them in specific cell types and at varying points during cell differentiation. Proper regulation of DNA methylation is critical for normal cell function. An important class of writers is the DNA methyl transferases (DNMTs). DNMTs are responsible both for establishing DNA methylation patterns as well as maintaining them during successive rounds of DNA replication. DNMTs are often mutated in cancers and the precise mechanisms for how DNMTs promote gene-specific DNA methylation in healthy cells are not well understood. Recent evidence has suggested that RNA plays important roles in regulating epigenetic marks. For example, several lncRNAs have been shown to interacts with DNMTs, primarily with the maintenance methylase DNMT1. In this extension proposal I aim to investigate the regulation of DNMT1 by RNAs. I will initially aim to elucidate the molecular basis for how RNA interacts with DNMT1 by uncovering core structural features required for DNMT1 binding. I will follow up on the preliminary data I have gathered during my K00 funding which includes uncovering a high affinity of DNMT1 for GU-rich RNA. Additionally, I have found that DNMT1 binds with high affinity to its own mRNA, specifically it’s 3’ UTR, which interestingly is also very GU-rich. I will determine the nature of this specific interaction by performing Cryo-EM on a DNMT1/GU-rich RNA complex together with additional biochemical assays to better understand the binding preference of DNMT1 for various RNAs. Together, this proposal will help elucidate how DNA methylation is misregulated in cancer and will additionally provide information about how RNAs help regulate this important process. Gaining an understanding of the molecular basis for how RNAs interact with DNMT1 may provide structural targets for use in the development of tissue-specific cancer therapeutics.