Barrier Insulators in Erythropoiesis

描述(由申请人提供)：屏障绝缘体创建了一种“屏障”，以防止异染色质介导的基因沉默，这对于调节正常发育和分化中细胞类型的特定基因表达至关重要。屏障绝缘体功能的扰动经常发生在与散发性和遗传性遗传病或癌症状态相关的染色体易位中，导致这些疾病特有的基因表达的变化。脊椎动物屏障元件的结构和功能还知之甚少。该项目的总体目标是定义和表征在红细胞生成过程中控制基因表达的屏障绝缘体。目标一的目标是确定与人类红系细胞中的功能性屏障绝缘体相关的共同调控信号。这些研究提出的假设是，通过染色质结构识别的细胞型特定屏障绝缘体存在共同的调控信号，蛋白质与组蛋白甲基转移酶和乙酰转移酶活性的结合，ATP依赖的核小体重塑活性，以及其他调控功能。这一目标将最先进的高通量基因组技术与功能研究相结合。在确定屏障绝缘体并进行功能验证后，将整合和分析组蛋白结构、调节蛋白结合和基因组组织。目标二的目的是解决这样一种假设，即Usf(上游刺激因子)蛋白招募与激活组蛋白修饰相关的酶和其他蛋白质，以阻止导致屏障绝缘体中基因沉默相关染色质变化扩散的机制(S)。这一假设是基于对鸡β珠蛋白基因簇中的HS4屏障绝缘体的研究和来自人类红系细胞的初步数据。将评估USF在造血干细胞、祖细胞和红细胞中的结合，并将其与基因组组织、组蛋白结构和调节蛋白结合结合起来。第三个目标是描述红系细胞中与屏障绝缘体相关的多蛋白复合体，解决这些复合体含有共同功能蛋白的假设，包括组蛋白甲基转移酶和乙酰基转移酶、核小体重塑蛋白和其他关键调控蛋白。由于丰度低、动态和上下文相关的组成以及识别复杂成分的技术困难，对多蛋白复合体基因表达调控程序的全面分析一直受到阻碍。为了克服这些障碍，将使用最先进的定量蛋白质组学技术对介导屏障绝缘体功能的多蛋白复合体进行表征。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，然后进行验证研究。总之，这些研究将为控制基因表达的关键过程提供新的见解，并最终将导致对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的全面理解。 与公共卫生相关：这项提议的目标是了解一种知之甚少但却很重要的遗传因素是如何在正确的时间在正确的地点开启和关闭基因的。当这一元素不起作用时，细胞可能不起作用，就像出生缺陷的孩子一样，或者可能会失去控制，就像癌症一样。了解这一因素可能会为我们提供有关某些疾病如何发生的重要信息，并可能为如何治疗这些疾病提供想法。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to understand how a poorly understood, yet important genetic element that turns genes on and off at the right time in the right place works. When this element doesn't work, cells may not work, as occurs in children born with birth defects, or may grow out of control, as occurs in cancer. Understanding this element may provide us with important information on how some diseases occur and may provide ideas on how to treat them.