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: 7405680
• 负责人: Roger Bancroft Deal
• 金额: $ 4.68万
• 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/7405680
• 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; Histones; Individual; Knowledge; Label; Lead; Ligase; Malignant Neoplasms; Mediating; Membrane Proteins; Methods; Modification; 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 type; chromatin immunoprecipitation; env Gene Products; epigenomics; 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(申请人提供)：体内的特殊细胞类型是由未分化的细胞通过染色质介导的基因组重编程过程产生的，由此建立并在表观遗传学上维持每种细胞类型特有的基因表达模式。然而，由于分离研究单个细胞类型的固有技术困难，我们对细胞分化机制的了解仍然非常有限。这项工作的目的是建立一种简单且广泛适用的方法，从特定类型的细胞中分离RNA和染色质，并利用该系统来研究拟南芥根的两种表皮细胞类型(毛细胞和非毛细胞)是由共同的祖细胞产生的。这些目标将通过以下具体目标来实现：(1)开发一种基于亲和力的方法，从特定类型的细胞中分离出细胞核。将大肠杆菌生物素连接酶(BIRA)与含有BIRA识别肽(BLRP)的核膜蛋白共表达，将产生生物素标记的核，可以使用链霉亲和素包裹的珠子从细胞提取物中分离出生物素标记的核。BIRA将被组成性地表达，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，在另一个单独的品系中在非毛细胞中表达。(2)分离毛细胞和非毛细胞核，用于全基因组表达分析和染色质免疫沉淀-微阵列(ChLP-CHIP)分析每种细胞类型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的转录组的定性和定量描述，并将揭示在一种或另一种类型中唯一表达的基因，而ChlP-ChIP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)使用反向遗传学来确定每种细胞类型所需的基因。通过操纵仅在一种或另一种细胞类型中表达的基因，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中心问题，而且与理解癌症的发展有关。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保持其特化状态提供一般的见解。这项研究中开发的技术也可以直接应用于研究易受癌变影响的细胞中的基因活性，以及从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，为开发新的治疗方法铺平道路。