Identification of a Molecular Signature for Barrier Insulators

描述(申请人提供)：基因激活和沉默控制在染色质水平。转录沉默的异染色质区域是动态的，如果没有积极的干预，就会扩散到常染色质区域，导致基因沉默。屏障绝缘元件的功能是维持常染色质和异染色质之间的边界，并且对于维持高阶染色质结构以调节适当的基因表达模式是必不可少的。为了了解我们的基因组是如何被调控的，对屏障绝缘体的彻底了解是必要的。这一关键调控机制的扰动导致了遗传性遗传病和癌症状态下的染色体易位后观察到的基因表达异常。此外，破坏屏障绝缘体功能的突变与遗传性溶血性贫血有关。这些基因表达异常的机制尚不清楚，因为人们对哺乳动物屏障绝缘体的结构和功能知之甚少。这项建议的目标是描述人类细胞中屏障绝缘体元件的位置、结构和功能。本提案的具体目标1的目标是确定与阻挡绝缘体元件相关的分子特征。我们假设这个信号将由USF蛋白及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，H3K4me2)组成。我们将利用染色质免疫沉淀和超高通量SolexA测序(CHIP-SEQ)来创建原代有核人类红系细胞中屏障绝缘物相关因子结合和染色质结构的全基因组图谱。将识别和选择屏障蛋白共占区域，并进行位置效应差异(PEV)分析，以确认这些区域代表功能性屏障绝缘体。这项建议的第二个具体目标是通过详细描述先前确定的一组潜在的屏障绝缘体的功能特征，获得对哺乳动物细胞中屏障绝缘体功能的全面了解，这些潜在屏障绝缘体位于红细胞膜蛋白基因的焦点组中。我们假设屏障绝缘体的活性是细胞类型特异性的，并且USF蛋白是绝缘体功能所必需的。为了深入了解绝缘子结构和功能的细胞类型特异性，候选屏障绝缘子将在红系和非红系细胞中进行定量芯片分析和PEV分析，并将在转基因小鼠模型中详细研究选定的位置。我们还将使用shRNA敲除USF蛋白来确定它们是否对哺乳动物细胞的屏障绝缘体功能是必要的。将全基因组技术和详细的功能分析相结合应用于屏障绝缘体的研究，将为这一基因调控的基本机制提供有价值的机械见解。

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.