Identification of a Molecular Signature for Barrier Insulators

势垒绝缘体分子特征的识别

基本信息

批准号：
8217248
负责人：
LAURIE A. STEINER
金额：
$ 14.77万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-02-01 至 2014-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8217248
关键词：
Acetyltransferase Architecture Area Binding Biological Assay Biological Models Cancerous Cells ChIP-seq Chickens Chromatin Chromatin Structure Chromosomal translocation Complex Coupled DNA DNA Binding DNA Sequence Data EP300 gene Elements Enhancers Ensure Erythrocyte Membrane Erythroid Cells Euchromatin Focus Groups Gene Activation Gene Expression Gene Expression Profile Gene Expression Regulation Gene Silencing Genes Genetic Genome Globin Goals Health Hemolytic Anemia Hereditary Disease Heterochromatin Higher Order Chromatin Structure Human Inherited Insulator Elements Intervention Laboratories Link Location Malignant Neoplasms Mammalian Cell Maps Mediating Membrane Protein Gene Methyltransferase Modeling Molecular Molecular Profiling Mutation PCAF gene Pattern Play Population Positioning Attribute Protein Binding Proteins Recruitment Activity Regulation Regulatory Element Reporter Genes Research Role Site Specificity Spectrin Structure Syndrome Technology Tissue Differentiation Tissues Transgenic Mice base cell type chromatin immunoprecipitation chromatin modification genetic regulatory protein genome-wide histone acetyltransferase histone methyltransferase human disease insight knock-down mouse model prospective protein 4.1 small hairpin RNA

项目摘要

DESCRIPTION (provided by applicant): Gene activation and silencing are controlled at the level of chromatin. Regions of transcriptionally silent heterochromatin are dynamic, and without active intervention, will spread into areas of euchromatin resulting in gene silencing. Barrier insulator elements function to maintain the boundaries between euchromatin and heterochromatin and are essential for maintaining higher order chromatin structure to regulate appropriate patterns of gene expression. To understand how our genome is regulated, a thorough understanding of barriers insulators is necessary. Perturbations in this critical regulatory mechanism contribute to the abnormalities in gene expression observed following chromosomal translocations in both inherited genetic disease and cancerous states. In addition, mutations disrupting barrier insulator function have been linked to inherited hemolytic anemia. The mechanisms underlying these aberrations in gene expression are unclear, because little is known about the structure and function of mammalian barrier insulators. The goal of this proposal is to characterize the location, structure, and function of barrier insulator elements in human cells. The goal of specific aim 1 of this proposal is to identify a molecular signature associated with barrier insulator elements. We hypothesize that this signature will be composed of the USF proteins, their associated histone methyltransferases (PRMT1, PRMT4, SET7/9), and acetyltransferases (CBP, P300, PCF), as well as an active chromatin structure (H3Ac, H4Ac, H3K4me2). We will utilize chromatin immunoprecipitation coupled with ultrahigh throughput Solexa sequencing (ChIP-seq) to create genome-wide maps of barrier insulator- associated factor binding and chromatin architecture in primary nucleated human erythoid cells. Regions of barrier protein co-occupancy will be identified and selected sites subjected to position effect variegation (PEV) assays to confirm that these regions represent functional barrier insulators. The goal of the second specific aim of this proposal is to gain a comprehensive understanding of barrier insulator function in mammalian cells through detailed functional characterization of a previously identified group of potential barrier insulators, located in a focus group of erythrocyte membrane protein genes. We hypothesize that barrier insulator activity is cell-type specific and that the USF proteins are necessary for insulator function. To gain insights into the cell- type specificity of insulator structure and function, candidate barrier insulators will be subjected to quantitative ChIP analyses and PEV assays in both erythroid and non-erythroid cells, and selected sites will be studied in detail in transgenic mouse models. We will also employ shRNA knock-down of the USF proteins to determine if they are necessary for barrier insulator function in mammalian cells. The combination of genome-wide technologies and detailed functional analyses applied to the study of barrier insulators will provide valuable mechanistic insights into this fundamental mechanism of gene regulation. PUBLIC HEALTH RELEVANCE: Barrier insulator elements are an important form of genetic regulation, which are not well understood in mammalian cells. Disruptions in these regulatory elements contribute to the abnormal gene expression seen in some cancers and inherited genetic syndromes. The goal of this application is to study the location, structure, and function of barrier insulators in mammalian cells, so that we can better understand this fundamental mechanism of gene regulation.

描述（由申请人提供）：基因激活和沉默在染色质水平上受到控制。转录静音异染色质的区域是动态的，如果没有主动干预，将扩散到斑塑素的区域，从而导致基因沉默。屏障绝缘子元件的功能以维持斑塑素和异染色质之间的边界，对于维持高阶染色质结构以调节适当的基因表达模式至关重要。要了解我们的基因组的调节方式，必须对障碍物绝缘子有透彻的理解。这种关键调节机制的扰动有助于在遗传遗传疾病和癌变状态下染色体易位后观察到的基因表达异常。另外，破坏屏障绝缘子功能的突变已与遗传性溶血性贫血有关。这些基因表达中这些像差的机制尚不清楚，因为对哺乳动物屏障绝缘子的结构和功能知之甚少。该建议的目的是表征人类细胞中屏障绝缘子元件的位置，结构和功能。该提案的特定目的1的目的是确定与屏障绝缘体元件相关的分子签名。我们假设该特征将由USF蛋白，其相关的组蛋白甲基转移酶（PRMT1，PRMT4，SET7/9）和乙酰基转移酶（CBP，P300，PCF）以及活性染色质结构（H3AC，H4AC，H4AC，H3K4ME2）。我们将利用染色质免疫沉淀以及超高吞吐量的solexa测序（CHIP-SEQ）在原代成核的人类红细胞细胞中创建屏障绝缘因子结合和染色质结构的全基因组图。将确定屏障蛋白联合占领的区域，并选择受到位置效应杂色（PEV）测定的选定位点，以确认这些区域代表功能性屏障绝缘体。该提案的第二个特定目的的目的是通过详细识别的型潜在屏障绝缘子组的详细功能表征在哺乳动物细胞中的屏障绝缘体功能中进行全面了解，该组位于红细胞膜蛋白基因的焦点组中。我们假设屏障绝缘体活性是细胞类型的特异性，并且USF蛋白对于绝缘体功能是必需的。为了洞悉绝缘体结构和功能的细胞类型特异性，候选屏障绝缘子将在红细胞和非果皮细胞中进行定量芯片分析和PEV分析，并将以转基因小鼠模型进行详细研究。我们还将采用USF蛋白的shRNA敲除，以确定它们在哺乳动物细胞中是否需要屏障绝缘体功能。全基因组技术和应用于屏障绝缘体研究的详细功能分析的组合将为这种基因调节的基本机制提供宝贵的机械见解。公共卫生相关性：障碍绝缘因子是遗传调节的一种重要形式，在哺乳动物细胞中尚不清楚。这些调节元件中的破坏有助于在某些癌症和遗传综合征中看到的异常基因表达。该应用的目的是研究哺乳动物细胞中屏障绝缘子的位置，结构和功能，以便我们可以更好地理解这种基因调节的基本机制。