Barrier Insulators in Erythropoiesis

项目负责人/主要研究者（最后，第一，中间）：Gallagher，帕特里克G。 项目摘要-摘要 红细胞生成中的屏障绝缘体 屏障绝缘体创造了一个“屏障”，以防止异染色质介导的基因沉默，这对 在正常发育和分化中调节细胞类型特异性基因表达。摄动 屏障绝缘子功能，这经常发生在染色体易位与散发性和 遗传性遗传疾病或癌症状态，有助于改变基因表达特征， 了以下条件脊椎动物屏障元件的结构和功能知之甚少。整体 该项目的目的是定义和表征控制基因表达的屏障绝缘体， 红细胞生成目标一的目标是识别与功能相关的共同监管签名， 人类红细胞中的屏障绝缘体。这些研究解决了一个假设，即有一个共同的 可被染色质结构识别的细胞类型特异性屏障绝缘子的调节信号， 具有组蛋白甲基转移酶和乙酰转移酶活性的蛋白质，ATP依赖性核小体重塑 活动和其他监管职能。这一目标结合了最先进的高通量基因组 技术与功能研究。在屏障绝缘子被识别和功能验证之后， 组蛋白结构，调节蛋白结合和基因组组织将被整合和分析。的 目的二的目标是解决USF（上游刺激因子）蛋白募集酶的假设 和其他与激活组蛋白修饰相关的蛋白质，以阻断导致 在屏障绝缘体中基因沉默相关染色质变化的扩散。这个假设是基于 来自β珠蛋白基因簇的鸡HS 4屏障绝缘子的研究和来自人类的初步数据 红系细胞将评估造血干细胞和祖细胞以及成红细胞中的USF结合， 整合了基因组组织、组蛋白结构和调节蛋白结合。第三个目标 目的是表征红系细胞中与屏障绝缘体相关的多蛋白复合物， 解决了这些复合物包含共同功能的蛋白质的假设，包括组蛋白 甲基转移酶和乙酰转移酶，核小体重塑蛋白，以及其他重要的调节蛋白。 proteins.对基因表达的多蛋白复合物调控程序的综合分析， 低丰度，动态和上下文相关的组成，以及技术困难， 识别复杂的成分。为了克服这些障碍，多蛋白复合物的表征 介导屏障绝缘子功能将使用最先进的定量蛋白质组学技术进行。 这种方法结合了体内稳定同位素标记蛋白质、集成质谱和 计算平台，然后进行验证研究。总之，这些研究将提供新的见解， 控制基因表达的关键过程，并最终导致对基因表达的全面理解。 在细胞生长和发育过程中控制特定基因表达程序的调节相互作用。 PHS 398/2590（Rev.06/09）

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