Barrier Insulators in Erythropoiesis

项目主任/首席调查员(最后、第一、中间)：加拉格尔，帕特里克·G 项目摘要-摘要 屏障绝缘子在红细胞生成中的应用 屏障绝缘体创建了一道“屏障”，以防止异染色质介导的基因沉默，这对 细胞类型特异性基因在正常发育和分化中的表达调控。摄动 屏障绝缘体功能，经常发生在染色体易位中，与散发性和 遗传性遗传性疾病或癌症状态，会导致基因表达特征的变化 这些条件。脊椎动物屏障元件的结构和功能还知之甚少。整体而言 该项目的目的是定义和表征控制基因表达的屏障绝缘体。 红血球生成。目标一目标是确定与功能相关的共同监管签名 人类红系细胞中的屏障绝缘体。这些研究解决了这样一种假设，即有一个共同的 可通过染色质体系结构识别的单元型特殊屏障绝缘子的规范签名 具有组蛋白甲基转移酶和乙酰转移酶活性的蛋白质与ATP依赖的核小体重塑 活动，以及其他监管职能。这一目标结合了最先进的高通量基因组 具有功能研究的技术。在已经识别并在功能上验证了阻挡绝缘体之后， 组蛋白结构、调节蛋白结合和基因组组织将被整合和分析。这个 目标二的目标是解决USF(上游刺激因子)蛋白质招募酶的假设。 和其他与激活组蛋白修饰相关的蛋白质来阻止导致 屏障绝缘体中与基因沉默相关的染色质变化的传播。这一假设是基于 鸡β珠蛋白基因簇中HS4屏障绝缘子的研究和人类的初步数据 红系细胞。将评估USF在造血干细胞、祖细胞和红细胞中的结合情况，并 整合了基因组组织、组蛋白结构和调节蛋白结合。第三届奥运会的目标 目的是表征红系细胞中与屏障绝缘体相关的多蛋白复合体， 说明这些复合体含有包括组蛋白在内的具有共同功能的蛋白质的假设 甲基转移酶和乙酰转移酶、核小体重塑蛋白和其他关键调控蛋白 蛋白质。多蛋白复合体基因表达调控程序的综合分析 由于丰度低、动态和上下文相关的组成，以及 识别复杂的成分。为了克服这些障碍，多蛋白复合体的表征 中介屏障绝缘体功能将使用最先进的定量蛋白质组学技术来执行。 这种方法结合了体内稳定同位素标记蛋白质、集成质谱学和 计算平台，随后进行验证研究。总而言之，这些研究将为我们提供对 控制基因表达的关键过程，最终将导致对 在细胞生长和发育过程中控制特定基因表达程序的调控相互作用。 PHS 398/2590(06/09版)页面续格式页面

Program Director/Principal Investigator (Last, First, Middle): Gallagher, Patrick G. Project Summary - Abstract Barrier Insulators in Erythropoiesis Barrier insulators create a "barrier" to protect against heterochromatin-mediated gene silencing, critical for regulation of cell-type specific gene expression in normal development and differentiation. Perturbation of barrier insulator function, which frequently occurs in chromosomal translocations associated with sporadic and inherited genetic disease or cancerous states, contributes to alterations in gene expression characteristic of these conditions. The structure and function of vertebrate barrier elements are poorly understood. The overall aims of this project are to define and characterize barrier insulators controlling gene expression during erythropoiesis. The goal of aim one is to identify a common regulatory signature associated with functional barrier insulators in human erythroid cells. These studies address the hypothesis that there is a common regulatory signature for cell-type specific barrier insulators recognizable by chromatin architecture, binding of proteins with histone methyltransferase and acetyltransferase activity, ATP-dependent nucleosome remodeling activity, and other regulatory functions. This aim combines state of the art high throughput genomic technologies with functional studies. After barrier insulators have been identified and functionally validated, histone architecture, regulatory protein binding, and genomic organization will be integrated and analyzed. The goal of aim two is to address the hypothesis that USF (upstream stimulatory factor) proteins recruit enzymes and other proteins associated with activating histone modifications to block the mechanism(s) that lead to spreading of gene-silencing associated chromatin changes in barrier insulators. This hypothesis is based on studies of the chicken HS4 barrier insulator from the beta globin gene cluster and preliminary data from human erythroid cells. USF binding in hematopoietic stem and progenitor cells and erythroblasts will be assessed and integrated with genomic organization, histone architecture, and regulatory protein binding. The goal of the third aim is to characterize the multiprotein complexes associated with barrier insulators in erythroid cells, addressing the hypothesis that these complexes contain proteins of common function, including histone methyltransferases and acetyltransferases, nucleosomal remodeling proteins, and other critical regulatory proteins. Comprehensive analyses of multiprotein complexes regulating programs of gene expression have been hampered by low abundance, dynamic and context-dependent composition, and technologic difficulties in identifying complex constituents. To overcome these hurdles, characterization of the multiprotein complexes mediating barrier insulator function will be performed using state-of-the-art, quantitative proteomics techniques. This approach combines protein labeling by stable isotopes in vivo, integrated mass spectrometry and computational platforms, followed by validation studies. Together, these studies will provide novel insight into a critical process controlling gene expression and will ultimately lead to a comprehensive understanding of the regulatory interactions that control specific gene expression programs during cell growth and development. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page