Measuring Methylation Kinetics in Cancer Cells: Computations and Experiments

描述(由申请人提供)：甲基化是一种基因沉默的表观遗传学机制，被发现在癌症进展中起重要作用。许多肿瘤抑制基因被认为是甲基化事件失活的。因此，研究甲基化的过程可以促进我们对癌症发生的理解。我们建议开发和实施计算和实验方法来测量癌细胞中的从头甲基化动力学。我们的总体目标是量化具有不同遗传背景的细胞系中启动子甲基化的速率。 我们将构建数学模型，描述在体外指数级生长的细胞群体中的启动子甲基化。关于甲基化过程的几个假设是不确定的；因此，我们将考虑各种不同的模型。测试这些模型将允许我们拒绝某些假设，并确定哪个模型与数据最一致。 我们将进行体外实验，以量化几种癌细胞系的从头甲基化过程。我们将通过使用COBRA和焦磷酸测序技术获得菌落大小的时间序列测量以及甲基化数据。然后，我们将使用实验数据来区分哪些模型是不正确的，哪些模型与数据最一致。这将阐明甲基化过程背后的机制。然后，我们将使用实验验证的最佳拟合模型来计算具有不同遗传背景的细胞中特定位置和平均的启动子甲基化速率。 实验室总结：这个项目将帮助我们更好地了解由于不适当的DNA甲基化而导致的结肠癌发展的速度/速率，这是一种在大多数人类癌症中普遍存在的机制。由于DNA甲基化是一个可逆的过程，更好地了解这些患者的癌症发展速度将有助于及时制定对癌症预防有影响的甲基化逆转疗法。项目说明： 癌症的发生和发展涉及细胞的遗传和表观遗传修饰。虽然遗传修饰(突变)已经被详细研究，但表观遗传变化(甲基化)还不是很清楚，所以我们建议研究癌症基因启动子的甲基化过程。通过测试和剔除各种数学模型，我们将发现哪些机制是这一过程的关键，并使用最佳拟合模型来测量不同细胞系的实际甲基化速率。

DESCRIPTION (provided by applicant): Methylation is an epigenetic mechanism of gene silencing found to be important in cancer progression. Many tumor suppressor genes are believed to be inactivated by methylation events. Therefore studying the process of methylation can advance our understanding of carcinogenesis. We propose to develop and implement computational and experimental approaches to measuring de novo methylation kinetics in cancer cells. Our overall aim is to quantify the rate of promoter methylation in cell lines with different genetic backgrounds. We will construct mathematical models which describe promoter methylation in a population of cells growing exponentially in vitro. Several assumptions regarding the methylation process are uncertain; therefore, we will consider a variety of different models. Testing these models will allow us to reject certain assumptions, and to determine which model is most consistent with data. We will conduct in vitro experiments to quantify the process of de novo methylation in several cancer cell lines. We will obtain time-series measurements of the colony size as well as methylation data, by using COBRA and pyrosequencing techniques. We will then use data from the experiments in order to distinguish which models are incorrect, and which model is most consistent with data. This will shed light on the mechanisms which underlie the methylation process. We will then use the experimentally validated, best-fitting model in order to calculate the site-specific and average rates of promoter methylation in cells with different genetic backgrounds. Lab summary: This project would help us better understand the speed/rate of colon cancer development that happens as a result of inappropriate DNA methylation, a mechanism that is prevalent in most human cancers. Since DNA methylation is a reversible process, a better knowledge of rate of cancer development in these patients would allow timely institution of methylation-reversing therapies that have implications for cancer prevention.Project Narrative: The initiation and progression of cancers involves both genetic and epigenetic modifications of cells. While genetic modifications (mutations) have been studied in detail, epigenetic changes (methylation) are not well understood, so we propose to study the methylation process of cancer gene promoters. By testing and rejecting various mathematical models we will find which mechanisms are key to the process, and use the best-fitting model to measure the actual methylation rate in different cell lines.