The dynamics of DNA methylation in normal and cancer cells

描述（由申请人提供）：哺乳动物DNA甲基化的基因组模式在基因调控和染色质组织中起着关键作用。DNA甲基化在多种细胞功能和病理中都很重要，包括组织特异性基因表达、细胞分化、基因组印记、X染色体失活、染色质结构调节、衰老和致癌。CG甲基化是哺乳动物基因组中最常见的甲基化类型，它由称为DNMT1的维持性DNA甲基转移酶维持，该酶作用于复制分叉处新合成的DNA，并显示出对半甲基化CG位点的偏好。UHRF1含有一个甲基DNA结合域，显示出对半甲基化CG位点的强优先结合。DNMT1部分通过与增殖细胞核抗原（PCNA）的相互作用，部分通过与UHRF1的直接相互作用被招募到复制病灶。在正常和转化的癌细胞中，甲基化DNA是如何维持在基因沉默位点的，目前还相对不清楚。UHRF1通过其SRA结构域结合半甲基化DNA；然而，目前尚不清楚UHRF1的各种其他结构域在维持DNA甲基化中起什么作用。我们的初步数据揭示了DNMT1上一个先前未被识别的甲基化位点。我们相信这可能是调节维持性DNA甲基化的另一种机制。该项目的目标是更详细地了解甲基化DNA是如何在正常和转化癌细胞的基因沉默位点维持的。UHRF1泛素样结构域、PHD、SRA和RING finger motif突变，靶向破坏UHRF1将在小鼠胚胎干（ES）细胞中稳定表达。将采用各种分析来帮助阐明这些结构域在维持DNA甲基化中所起的作用。与未甲基化肽相比，合成的DNMT1甲基化肽也将在体外测试与UHRF1 PHD结构域的直接结合。甲基化DNMT1的功能也将通过敲除和敲除DNMT1甲基化所需的甲基转移酶来检测。最后，将在小鼠胚胎干细胞中进行shRNA筛选，以鉴定DNA甲基化所需的其他基因。越来越多的证据表明，到目前为止，在大多数癌症中，DNA甲基化组都经历了特征性的变化。这与癌变机制高度相关，因为错误调节的DNA甲基化模式可以促进许多癌细胞类型的异常基因表达和转移特性。这些研究的长期目标是确定DNA甲基化在正常细胞向癌症表型的转化中所起的作用。

DESCRIPTION (provided by applicant): Genomic patterns of DNA methylation in mammals play a critical role in gene regulation and chromatin organization. DNA methylation is important in a diverse range of cellular functions and pathologies, including tissue-specific gene expression, cell differentiation, genomic imprinting, X chromosome inactivation, regulation of chromatin structure, aging, and carcinogenesis. CG methylation is the most common type found in mammalian genomes and it is maintained by the maintenance DNA methyltransferase called DNMT1, which acts on newly synthesized DNA at replication forks and shows a preference for hemimethylated CG sites. UHRF1 contains a methyl DNA binding domain that shows strong preferential binding to hemimethylated CG sites. DNMT1 is recruited to replication foci in part through its interaction with proliferating cell nuclear antigen (PCNA), and in part by directly interacting with UHRF1. How methylated DNA is maintained at sites of gene silencing in normal and transformed cancer cells is still relatively unclear. UHRF1 binds hemimethylated DNA through its SRA domain; however, it is unclear what roles the various other domains of UHRF1 play in maintaining DNA methylation. Our preliminary data has revealed a previously unidentified site of methylation on DNMT1. We believe this might be another mechanism for regulating the maintenance DNA methylation. The goal of this project is to understand in greater detail how methylated DNA is maintained at sites of gene silencing in normal and transformed cancer cells. UHRF1 with mutations of the ubiquitin like domain, PHD, SRA, and the RING finger motif, respectively, will be stably expressed in murine embryonic stem (ES) cells with a targeted disruption of UHRF1. Various assays will be employed to help elucidate the roles these domains play in maintaining DNA methylation. Synthesized methylated peptides of DNMT1 will also be tested for direct binding to the UHRF1 PHD domain in vitro compared to unmethylated peptides. The function of methylated DNMT1 will also be examined by performing knockout and knockdown of the methyltransferase required for methylating DNMT1. Finally, an shRNA screen in murine ES cells will be developed to identify other genes required for DNA methylation. There is growing evidence that the DNA methylome undergoes characteristic changes in the majority of cancers examined thus far. This is highly relevant to mechanisms of carcinogenesis, since mis-regulated DNA methylation patterns can promote abnormal gene expression and metastatic properties of numerous cancer cell types. The long-term goal of these studies is to determine what role DNA methylation is playing in the transformation of a normal cell to a cancer phenotype.