A new epigenome profiling method for the study of cell fate specification

用于研究细胞命运规范的新表观基因组分析方法

• 批准号: 7555910
• 负责人: Roger Bancroft Deal
• 金额: $ 5.01万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7555910
• 关键词: Affinity; Biotin; Cancerous; Cell Extracts; Cell Nucleus; Cells; Chromatin; Development; Developmental Biology; E coli birA protein; Ectopic Expression; Enzymes; Epigenetic Process; Gene Expression; Gene Expression Profile; Genes; Genome; Goals; Hair Cells; Individual; Knowledge; Label; Lead; Ligase; Malignant Neoplasms; Mediating; Membrane Proteins; Methods; Mouse-ear Cress; Nuclear Envelope; Pattern; Peptides; Plant Roots; Process; RNA; Regulator Genes; Research; Specific qualifier value; Stem cells; Streptavidin; System; Technology; Transgenic Organisms; Tumor-Derived; Undifferentiated; Whole Organism; Work; base; cell fate specification; cell type; chromatin immunoprecipitation; env Gene Products; epigenomics; genome-wide; histone modification; insight; mutant; positional cloning; research study

DESCRIPTION (provided by applicant): Specialized cell types in the body are produced from undifferentiated cells through a process of chromatin mediated genome reprogramming whereby the pattern of gene expression unique to each cell type is established and epigenetically maintained. However, because of inherent technical difficulties in studying individual cell types in isolation, our knowledge of the mechanisms of cellular differentiation remains extremely limited. The goals of the work proposed herein are to develop a simple and widely applicable method for the isolation of RNA and chromatin from specific cell types, and to use this system to investigate the mechanisms by which the two epidermal cell types of the Arabidopsis thaliana root (hair cells and nonhair cells) are generated from common progenitor cells. These goals will be achieved through the following specific aims: (1) Development of an affinity-based method for the isolation of nuclei from specific cell types. Coexpression of the E. coli biotin ligase enzyme (BirA) along with a nuclear envelope protein containing the BirA recognition peptide (BLRP) will produce biotin labeled nuclei that can be isolated from cell extracts using streptavidin-coated beads. BirA will be expressed constitutively, while the BLRP-tagged nuclear membrane protein will be expressed in root hair cells in one transgenic line, and in non-hair cells in a separate line. (2) Isolation of hair cell and non-hair cell nuclei for genome-wide expression analysis and chromatin immunoprecipitation-microarray (ChlP-chip) analysis of histone modifications throughout the genome in each cell type. Gene expression studies will provide a qualitative and quantitative description of the transcriptome of each cell type and will reveal the genes that are expressed exclusively in one type or the other, while ChlP-chip analysis will yield insight into the epigenetic processes underlying the transcriptional profile of each cell type. (3) Use reverse genetics to identify genes that are required for the specification of each cell type. By manipulating genes expressed exclusively in one cell type or the other, we can fully dissect how the hair cell and non-hair cell identities are specified. Understanding the mechanisms by which cell fate is established and maintained is not only a central problem in developmental biology, but is also relevant to understanding the development of cancer. RELEVANCE: This research will yield general insight into how specialized cells are generated from stem cells, and how they maintain their specialized state. The technology developed in this research can also be applied directly to the study of gene activity in cells that are susceptible to cancerous changes, and to tumors derived form those cells. In this way, the changes that lead to cancer can be determined, paving the way for the development of new therapies.

描述（由申请人提供）：体内的特化细胞类型是通过染色质介导的基因组重编程过程从未分化的细胞中产生的，通过该过程，每种细胞类型特有的基因表达模式被建立并在表观遗传学上得到维持。然而，由于分离研究单个细胞类型固有的技术困难，我们对细胞分化机制的了解仍然非常有限。本研究的目的是建立一种简单、可广泛应用的从特定细胞类型中分离RNA和染色质的方法，并利用该系统研究拟南芥根部两种表皮细胞（毛细胞和非毛细胞）从共同祖细胞中生成的机制。这些目标将通过以下具体目标实现：(1)发展一种基于亲和力的方法，从特定细胞类型中分离细胞核。大肠杆菌生物素连接酶（BirA）与含有BirA识别肽的核包膜蛋白（BLRP）的共表达将产生生物素标记的细胞核，可以使用链霉亲和素包被珠从细胞提取物中分离出来。BirA将组成表达，而blrp标记的核膜蛋白将在一个转基因品系的根毛细胞中表达，在另一个品系的非毛细胞中表达。(2)分离毛细胞和非毛细胞细胞核进行全基因组表达分析和染色质免疫沉淀微阵列（ChlP-chip）分析各组细胞基因组中的组蛋白修饰。基因表达研究将提供每种细胞类型转录组的定性和定量描述，并将揭示仅在一种类型或另一种类型中表达的基因，而chlp芯片分析将深入了解每种细胞类型转录谱背后的表观遗传过程。(3)利用反向遗传学鉴定每种细胞类型所需的基因。通过操纵在一种或另一种细胞类型中专门表达的基因，我们可以充分剖析毛细胞和非毛细胞身份是如何指定的。了解细胞命运建立和维持的机制不仅是发育生物学的核心问题，而且与了解癌症的发展有关。相关性：这项研究将对干细胞如何产生特化细胞以及它们如何维持其特化状态产生一般性见解。这项研究中开发的技术还可以直接应用于研究易发生癌变的细胞中的基因活性，以及这些细胞衍生的肿瘤。通过这种方式，可以确定导致癌症的变化，为开发新疗法铺平道路。