Mechanisms of De Novo Methylation in Cancer

项目摘要/摘要 这笔资金已资助了近30年，其目的是了解 DNA甲基化模式的建立和遗传机制以及开发可 可以干扰胞嘧啶甲基化并重新激活沉默的基因。这项研究导致了最近的 FDA批准两种DNA去甲基化药物(5-aza-CR和5-aza-cdr)用于治疗髓系疾病 发育不良综合征。在该项目的下一个五年期间，我们希望利用表观基因组学 分析以了解DNA甲基化模式是如何通过相互作用建立和维持的 DNA甲基转移酶和特定染色质成分之间的关系。为了做到这一点，我们开发了一个 定制NimbleGen阵列，可用于分析核小体、组蛋白修饰和DNA 正常细胞和转化细胞中1,800个转录起始点(TSS)的整合甲基化。在……里面 具体目标1，我们将利用平铺阵列来定位正常(PrECs)和转化后的核小体 前列腺癌细胞(PC3)。然后我们将确定如何从药理上干扰DNA甲基化 在PC3细胞中或在HCT116中从基因上讲，结肠癌细胞改变了组蛋白标记的分布，集中在 组蛋白H3-K27me3标记由多梳抑制复合体2(PRC2)应用。在具体目标2中，我们将 确定在DNA甲基化恢复到HCT116衍生物期间表观基因组是如何重组的 (DKO)，其中三个DNA甲基转移酶中的两个(DNMT1和Dnmt3b)已被遗传 被撞倒了。在具体目标3中，我们将跟进我们的新结果，这些结果表明 从头开始甲基转移酶DNMT3A和3B到核小体。我们希望确定这些酶是如何 与核小体相互作用，这样我们就可以理解特定的模式是如何建立的。在具体目标4中， 我们将继续探索开发比目前更稳定的DNA去甲基化药物 被FDA批准用于癌症治疗，并能够逆转异常DNA甲基化的组蛋白 修饰和核小体定位。这些目标的实现应该对我们的 了解癌症的表观遗传学，并与新的治疗和预防策略直接相关 癌症。

Project Summary/Abstract The objectives of this grant, which has been funded for almost 30 years, have been to understand the mechanisms for the establishment and inheritance of DNA methylation patterns and to develop drugs which can interfere with cytosine methylation and reactivate silenced genes. This research has led to the recent approval by the FDA of two DNA demethylating agents (5-aza-CR and 5-aza-CdR) for the treatment of myeloid dysplastic syndrome. In the next five year period of the project, we hope to take advantage of epigenomic analysis to understand how DNA methylation patterns are established and maintained by an interaction between DNA methyltransferases and specific chromatin components. To do this, we have developed a custom NimbleGen array allowing for the analyses of nucleosomes, histone modifications and DNA methylation in an integrated way at 1,800 transcription start sites (TSS) in normal and transformed cells. In Specific Aim 1, we will utilize the tiling array to map nucleosomes in both normal (PrECs) and transformed prostate cancer cells (PC3). We shall then determine how interfering with DNA methylation pharmacologically in PC3 cells or genetically in HCT116 colon cancer cells alters the distribution of histone marks focusing on the histone H3-K27me3 mark applied by the polycomb repressive complex 2 (PRC2). In Specific Aim 2, we shall determine how the epigenome is reorganized during the restoration of DNA methylation to HCT116 derivatives (DKO) in which two of the three DNA methyltransferases (DNMT1 and DNMT3B) have been genetically knocked down. In Specific Aim 3, we will follow-up on our new results which show the strong anchoring of the de novo methyltransferases DNMT3A and 3B to nucleosomes. We wish to determine how the enzymes interact with nucleosomes so that we can understand how specific patterns are established. In Specific Aim 4, we will continue our quest to develop DNA demethylating drugs which are more stable than those currently approved by the FDA for cancer treatment and which are able to reverse aberrant DNA methylation, histone modifications and nucleosome positioning. Achievement of these aims should have major impact in our understanding of the epigenetics of cancer and have direct relevance to new strategies to treat and prevent cancer.