Epigenomic profiling of histone turnover kinetics in mammalian cells

描述(由申请人提供)： 哺乳动物细胞中组蛋白周转动力学的表观基因组图谱我们已经开发出一种革命性的技术，CATCH-IT(用于标记的共价连接以捕获组蛋白并识别周转)，以直接测量核小体周转。Catch-IT方法包括用氨基酸类似物标记新合成的蛋白质，用生物素部分衍生，选择性地提取核小体核心颗粒，用链霉亲和素亲和纯化，以及提取DNA用于全基因组图谱。我们已经成功地获得了果蝇培养细胞的全基因组Catch-IT图谱，并利用这些数据来研究组蛋白周转与基本过程之间的关系，包括转录起始和延伸、表观遗传调节和复制起点的确定。在目前的提案中，我们的目标是将Catch-IT技术应用于哺乳动物，并调查一系列细胞类型和表观遗传过程中的核小体周转动力学。在目标1中，我们将把Catch-IT应用于多能干细胞和分化的早期步骤。在目标2中，我们将使用Burkitt淋巴瘤的E5-Myc小鼠模型和其他上皮细胞的小鼠模型，将Catch-IT应用于癌症研究。在目标3中，我们将应用Catch-IT来检测环境扰动的影响，包括类固醇激素处理、DNA损伤和热休克对组蛋白转换动力学的影响。由于Catch-IT不需要转基因品系、抗体或标签，它有可能成为了解染色质动力学的宝贵工具，以及监测与人类健康和疾病相关的表观遗传变化的通用系统。

DESCRIPTION (provided by applicant): Epigenomic profiling of histone turnover kinetics in mammalian cells We have developed a revolutionary technology, CATCH-IT (for Covalent Attachment of Tags to Capture Histones and Identify Turnover) to directly measure nucleosome turnover. The CATCH-IT method involves labeling of newly synthesized proteins with an amino acid analog, derivatization with a biotin moiety, selective extraction of nucleosome core particles, affinity purification with streptavidin, and extraction of DNA for genome-wide profiling. We have successfully obtained genome-wide CATCH-IT profiles for Drosophila cultured cells, and we have used these data to address the relationship between histone turnover and fundamental processes, including transcriptional initiation and elongation, epigenetic regulation and determination of replication origins. In the present proposal, we aim to apply CATCH-IT technology to mammals, and to survey nucleosome turnover dynamics in a range of cell types and epigenetic processes. In Aim 1, we will apply CATCH-IT to pluripotent stem cells and early steps in differentiation. In Aim 2, we will apply CATCH-IT to cancer studies, using the E5-Myc mouse model of Burkitt's lymphoma and other mouse models of epithelial cells. In Aim 3, we will apply CATCH-IT to examine the effects of environmental perturbation, including steroid hormone treatment, DNA damage and heat shock on histone turnover kinetics. As CATCH-IT does not require transgenic lines, antibodies or tags, it has the potential of becoming both an invaluable tool for understanding chromatin dynamics and a general system for monitoring epigenetic changes relevant to human health and disease.