Maintenance of Gene Expression

基因表达的维持

基本信息

批准号：
8371445
负责人：
ROBERT KINGSTON
金额：
$ 66.07万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-05-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8371445
关键词：
Address Area Binding Binding Sites Cell Culture Techniques Cells Chromatin Chromatin Structure Complex DNA Development Drosophila genome Drosophila genus Elements Family Functional RNA Funding Gene Expression Gene Silencing Genes Genetic Transcription Genome Goals Grant Histocompatibility Testing Histone H2A Histone H3 Histones Homologous Gene Human In Vitro Knowledge Lead Length Location Lysine Maintenance Mammals Maps Methods Methylation Mitosis Molecular Mus Nucleosomes Organism PRC1 Protein Play Polycomb Proteins Recruitment Activity Regulation Regulator Genes Reporter Repression Resolution Response Elements Role Site Structure System Techniques Technology Tertiary Protein Structure Testing Ursidae Family Work cell type design embryonic stem cell in vivo member mutant new technology protein complex protein function reconstitution research study scaffold technique development tool

项目摘要

DESCRIPTION (provided by applicant): For development of complex organisms to occur properly, the genes that determine cell type must be regulated both spatially and temporally. One key aspect of this regulation is the effective lineage-specific repression of master regulatory genes that must be maintained in an ¿off¿ status. This problem has been studied since the 1940s in Drosophila, which has led to the identification of a group of genes called the Polycomb- Group (PcG) after the founding member, the Polycomb gene. The PcG encodes proteins that form complexes that direct a repressed state. The central repressive complex in this family is called Polycomb Repressive Complex 1 (PRC1), and a second complex that methylates histones and helps target PcG function is called PRC2. PRC1 and PRC2 are targeted to appropriate locations by Polycomb Response Elements (PREs), which are usually about 1 kb in length and nucleosome depleted. This application describes experiments designed to understand function of the PcG system, with a focus on the role of nucleosome compaction, PRE function and the ability of non-coding RNAs to regulate this system. Three areas are addressed in three specific Aims in this application. Aim 1 examines the ability of a mammalian Polycomb protein called Cbx2 to compact nucleosomes in vitro and in cell culture. It also describes approaches to crystallize proteins involved in creating a compacted state with the goal of defining these mechanisms for Polycomb at this high level of resolution. Aim 2 explores the location and function of PREs in the human and mouse HOX clusters. Nucleosome depletion is used as one method to identify potential PRE sequences, which are then tested for function using reporter constructs. The hypothesis that the Jarid2 protein is generally involved in mammalian PRE function is tested using these PRE sequences. Aim 3 describes the development of a new technology to map the binding sites for ncRNAs. This technology will be used to test the hypothesis that specific ncRNAs are involved in targeting PcG function to specific loci. PUBLIC HEALTH RELEVANCE: We describe experiments to characterize the several aspects of the complex gene system, called the Polycomb-Group, that silences genes to maintain proper cell identity in mammals. We will characterize the protein domain and the molecular mechanism that creates a compacted chromatin structure perhaps causal to silencing, will identify DNA elements that target this system to specific genes, and will develop techniques to determine where non-coding RNAs involved in regulation function on the genome.

描述（由申请人提供）：为了使复杂生物体正常发育，决定细胞类型的基因必须在空间和时间上受到调节，这种调节的一个关键方面是对主调节的有效谱系特异性抑制。必须维持在 ¿关闭“关闭”自 20 世纪 40 年代以来，人们一直在果蝇中研究这个问题，这导致了一组称为 Polycomb 基因组 (PcG) 的基因的发现，该组基因以创始成员 Polycomb 基因的名字命名，该基因编码形成指导复合物的蛋白质。该家族中的中央抑制复合物称为 Polycomb 抑制复合物 1 (PRC1)，而第二种可甲基化组蛋白并帮助靶向 PcG 功能的复合物称为PRC1 和 PRC2 通过 Polycomb 响应元件 (PRE) 定位到适当的位置，其长度通常约为 1 kb，并且核小体已耗尽。本应用描述了旨在了解 PcG 系统功能的实验，重点关注的作用。本申请的三个具体目标涉及核小体压缩、PRE 功能和非编码 RNA 调节该系统的能力。它还描述了一种名为 Cbx2 的哺乳动物 Polycomb 蛋白在体外和细胞培养物中压缩核小体的能力，还描述了参与创建压缩状态的蛋白质结晶方法，目的是在 Aim 2 探索的这种高分辨率下定义 Polycomb 的这些机制。 PRE 在人和小鼠 HOX 簇中的位置和功能被用作识别潜在 PRE 序列的一种方法，然后使用报告构建体测试其功能。 Jarid2 蛋白通常参与使用这些 PRE 序列测试哺乳动物 PRE 功能。目标 3 描述了绘制 ncRNA 结合位点的新技术的开发，该技术将用于测试特定 ncRNA 参与将 PcG 功能靶向特定位点的假设。公共健康相关性：我们描述了描述复杂基因系统（称为 Polycomb 组）的几个方面的实验，该系统通过沉默基因来维持哺乳动物中正确的细胞身份。我们将描述蛋白质结构域和产生压缩染色质的分子机制。可能导致沉默的结构，将识别将该系统靶向特定基因的DNA元件，并将开发确定非编码RNA在基因组上参与调节功能的技术。