Project 2: Normal Cell Evolution

摘要：人体小复制单位的体细胞进化 该项目研究了正常生物进化的基本参数(突变、漂移和选择 人类和动物细胞。尽管MDS是体细胞进化的基础，但人们对此知之甚少 参数，因为它们很难在人类身上研究。我们将把进化生物学的方法转化为 进入系统生物学领域。我们将在不同的小复制单位(肠隐窝)中研究MDS。这个 将细胞划分为小的复制单位可以改变进化，因为选择和漂移 (随机信元周转)仅限于紧邻的信元。分析小复制基因的优势 单位是指在实验上，它们足够大，可以用传统方法测量，但又足够小， 详细模拟。表征复制单位中的体细胞进化可以更好地理解 肿瘤的进化是因为相邻细胞之间发生了选择或漂移。 我们将在现有发表的(柴田，格雷厄姆)人类的基础上，更好地描述墓穴中的MDS 地窖模拟。这些模拟已经推断出干细胞的数量和动态，基于 数据量较小。新的测序数据是一个更丰富的资源，因为更多的MDS参数 由突变(突变率和机制、DN/DS、乘客与司机、新抗原)编码 积累)。通过加密表观遗传和表达数据来扩充测序数据，以更充分地 描述正常的体细胞进化。我们将从40个不同的组织中抽取8个结肠和小肠隐窝 上了年纪的人。对于每个隐窝，我们将测量突变(全基因组测序)、表观遗传变化 (atac-seq)和表达式(纳米字符串)。我们还将测量APC+/-加密以确定 在看门人突变后，体细胞进化发生了变化。我们还将测量隐窝体细胞 老鼠和大象洞穴的进化以确定它们的进化是否不同。 我们还将测定57种不同哺乳动物成纤维细胞的DNA损伤和应激反应 物种。我们将通过扩增来确定同样57个哺乳动物细胞系的从头突变率 对单个成纤维细胞进行深度测序。我们将把突变率和癌症发病率联系起来。 项目1中确定的相同哺乳动物物种。最后，使用在 项目一，我们将对最有可能来自不同物种的前5个基因进行基因编辑实验 有助于他们抗癌能力的进化。 这些研究的意义在于更好地表征了基本的MDS演化参数。这个 物种研究将提供关于MDS参数是否“固定”或随年龄或年龄变化的观点 物种，确定哪些参数更适合进行干预。一个完整的系统生物学解决方案 对正常人体隐窝的研究将有助于进一步研究更大的体细胞组。该项目将铺设 为未来的工作奠定基础，通过基于进化的癌症解决方案影响患者护理。

Abstract: Somatic Cell Evolution in Small Human Replicative Units This Project studies fundamental parameters of evolution (mutation, drift, and selection "MDS") in normal human and animal cells. Although MDS underlies somatic cell evolution, very little is understood about these parameters because they are difficult to study in humans. We will translate methods from evolutionary biology into systems biology. We will study MDS in distinct, small replicative units (intestinal crypts). The compartmentalization of cells into small replicative units can modify evolution because selection and drift (random cell turnover) is limited to immediately adjacent cells. The advantages of analyzing small replicative units are that experimentally they large enough to measure with conventional methods yet small enough to simulate in detail. Characterizing somatic cell evolution in replicative units can lead to better understanding of tumor evolution because selection or drift occurs between neighboring cells. We will better characterize MDS in crypts based on our existing published (Shibata, Graham) human crypt simulations. These simulations have already inferred stem cell numbers and dynamics based on smaller amounts of data. The new sequencing data are a richer resource because more MDS parameters are encoded by mutations (mutation rates and mechanisms, dN/dS, passenger versus driver, neoantigen accumulation). The sequencing data is augmented by crypt epigenetic and expression data to more fully characterize normal somatic cell evolution. We will sample 8 colon and small intestinal crypts from 40 different aged individuals. For each crypt, we will measure mutations (whole genome sequencing), epigenetic alterations (ATAC-seq), and expression (NanoString). We will also measure APC+/- crypts to determine whether somatic cell evolution changes after a gatekeeper mutation. We will also measure crypt somatic cell evolution in mouse and elephant crypts to determine if their evolution differs. We will also determine the DNA damage and stress response in fibroblasts from 57 different mammalian species. We will determine de novo mutation rates across the same 57 mammalian cell lines through expansion of single fibroblast cells followed by deep sequencing. We will correlate mutation rates with cancer rates in the same mammalian species as determined in Project 1. Finally, using prioritized candidate gene lists generated in Project 1, we will perform gene editing experiments on the top 5 genes from different species most likely to contribute to their evolution of cancer resistance. The significance of these studies is a better characterization of basic MDS evolution parameters. The species studies will provide perspective on whether MDS parameters are "fixed" or can vary with age or species, identifying which parameters are more amenable for intervention. A complete systems biology solution of normal human crypts will facilitate further efforts with much larger groups of somatic cells. This Project will lay the groundwork for future work to impact patient care through evolutionary-based solutions to cancer.