DE NOVO METHYLTRANSFERASES AND CANCER

胞嘧啶碱基甲基化对DNA的修饰是一种基本的表观遗传机制，通过这种机制，一系列发育和细胞过程受到调节。已经证明，DNA甲基化对动物发育至关重要，因为小鼠体内组成性表达的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控、病毒感染抑制、基因组印迹和X染色体失活等方面发挥着重要作用。近年来，许多研究也将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的从头甲基化是一个发育调节的过程，在早期胚胎细胞中激活，但在成人组织中是惰性的。成人组织中新生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，一个新的DNA甲基转移酶基因家族，被称为Dnmt3a和Dnmt3b，已经从人和小鼠中克隆出来。小鼠基因在胚胎干细胞中表达，但在成年体细胞组织中表达较弱。为了研究Dnmt3甲基转移酶是如何被调节的，以及它们在小鼠发育过程中起什么作用，并确定DNA甲基化或Dnmt3表达的改变是否会导致癌症，我们将追求以下特定目标。目的1：利用基因靶向方法制备dnmt3缺陷小鼠，并对其发育缺陷进行表征。目的2：分析小鼠发育过程中Dnmt3的表达，并鉴定调节Dnmt3转录的顺式作用元件。目的3：确定内源性Dnmt3表达是否受致癌转化的诱导，以及Dnmt3转基因表达对裸鼠CpG岛新生甲基化、致癌转化和肿瘤形成的影响。目的4：评价转基因小鼠Dnmt3基因失活或异位表达对肿瘤进展或抑制的遗传影响。肠道肿瘤模型Min小鼠将用于测试Dnmt3a和Dnmt3b是否像Dnmt1一样在肠道息肉形成中起作用。本提案中描述的分子和遗传方法将揭示新生甲基化在发育和癌症中的生物学功能。

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.