DE NOVO METHYLTRANSFERASES AND CANCER

从头甲基转移酶与癌症

• 批准号: 6377347
• 负责人: EN LI
• 金额: $ 22.36万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6377347
• 关键词: CpG islands; DNA methylation; athymic mouse; carcinogenesis; developmental genetics; embryonic stem cell; gene expression; gene targeting; genetic transcription; genetically modified animals; immunocytochemistry; in situ hybridization; laboratory mouse; methyltransferase; neoplastic process; neoplastic transformation

DNA modification by methylation of the cytosine base is a fundamental epigenetic mechanism by which a range of developmental and cellular processes are regulated. It has been demonstrated that DNA methylation is essential for animal development, since inactivation of a constitutively expressed DNA methylation enzyme Dnmt1 in mice result in embryonic lethality. DNA methylation plays important roles in regulation of gene expression, suppression of viral infection, genomic imprinting and X chromosome inactivation. In recent years, numerous studies have also linked DNA methylation to various types of human cancer. However, the underlying mechanisms by which alterations in DNA methylation promotes cancer formation is large unknown. De novo methylation of unmodified DNA is a developmentally regulated process which is activated in early embryonic cells, but is inert in adult tissues. Aberrant activation of de novo methylation in adult tissues has been associated with transcriptional silencing of tumor suppressor genes in multiple tumors. Recently, a novel family of de novo DNA methyltransferase genes, termed Dnmt3a and Dnmt3b, have been cloned from both human and mouse. The mouse genes are expressed in embryonic stem cells, but weakly in adult somatic tissues. To investigate how Dnmt3 methyltransferases are regulated and what roles they play during mouse development, and to determine whether alterations in DNA methylation or Dnmt3 expression causes cancer, the following specific aims will be pursued. Aim 1: To generate Dnmt3-deficient mice using gene targeting methods and characterize their developmental defects. Aim 2: To analyze Dnmt3 expression during mouse development and identify cis-acting elements that regulate Dnmt3 transcription. Aim 3: To determine whether endogenous Dnmt3 expression is induced by oncogenic transformation, and the effect of Dnmt3 transgene expression on de novo methylation of CpG islands, oncogenic transformation, and tumor formation in nude mice. Aim 4: To evaluate the genetic effects of inactivation or ectopic expression of Dnmt3 in transgenic mice on tumor progression or suppression. The intestinal tumor model, the Min mice, will be used to test whether Dnmt3a and Dnmt3b have a role in intestinal polyp formation like Dnmt1. The molecular and genetic approaches described in this proposal will unravel the biological function of de novo methylation in development and cancer.

通过胞嘧啶碱基甲基化进行的 DNA 修饰是一种基本的表观遗传机制，通过它来调节一系列发育和细胞过程。 已经证明，DNA 甲基化对于动物发育至关重要，因为小鼠体内持续表达的 DNA 甲基化酶 Dnmt1 失活会导致胚胎死亡。 DNA甲基化在基因表达调节、病毒感染抑制、基因组印记和X染色体失活中发挥着重要作用。 近年来，大量研究也将 DNA 甲基化与各种类型的人类癌症联系起来。 然而，DNA 甲基化的改变促进癌症形成的潜在机制尚不清楚。未修饰 DNA 的从头甲基化是一种发育调控过程，在早期胚胎细胞中被激活，但在成体组织中呈惰性。 成人组织中从头甲基化的异常激活与多种肿瘤中肿瘤抑制基因的转录沉默有关。 最近，从人类和小鼠身上克隆了一个新的 DNA 甲基转移酶基因家族，称为 Dnmt3a 和 Dnmt3b。 小鼠基因在胚胎干细胞中表达，但在成体体组织中表达较弱。 为了研究 Dnmt3 甲基转移酶的调节方式及其在小鼠发育过程中发挥的作用，并确定 DNA 甲基化或 Dnmt3 表达的改变是否会导致癌症，我们将追求以下具体目标。 目标 1：利用基因打靶方法生成 Dnmt3 缺陷小鼠并表征其发育缺陷。 目标 2：分析小鼠发育过程中的 Dnmt3 表达并鉴定调节 Dnmt3 转录的顺式作用元件。 目的3：确定内源性Dnmt3表达是否由致癌转化诱导，以及Dnmt3转基因表达对CpG岛从头甲基化、致癌转化和裸鼠肿瘤形成的影响。 目标 4：评估转基因小鼠中 Dnmt3 失活或异位表达对肿瘤进展或抑制的遗传效应。 Min小鼠肠道肿瘤模型将用于测试Dnmt3a和Dnmt3b是否像Dnmt1一样在肠息肉形成中发挥作用。 该提案中描述的分子和遗传学方法将揭示从头甲基化在发育和癌症中的生物学功能。