Developmental Functions of SNR1 and the BRM Chromatin Remodeling Complex in Drosophila

果蝇中 SNR1 和 BRM 染色质重塑复合体的发育功能

• 批准号: 0221563
• 负责人: Andrew Dingwall
• 金额: $ 34.5万
• 依托单位: Syracuse University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0221563&HistoricalAwards=false
• 关键词: Developmental; Functions; SNR1; BRM; Chromatin

A fundamental challenge among all multicellular eukaryotes is coordinating the expression of critically important genes in different cells throughout development to produce a fully functional adult. This coordination is accomplished by short-range inductive signals between cells that rely on diffusible peptides and/or direct cell contacts to initiate intracellular signaling cascades that ultimately influence the expression of specific target genes. Chromatin has emerged as one of the primary obstacles with which the cell's transcription and replication machinery must contend. Factors necessary to transcribe or replicate DNA must gain access to regulatory sites that are packaged into nucleosomes and higher order structures. This is especially problematic when cells are in mitosis and chromatin is extremely condensed. Energy dependent chromatin remodeling complexes have evolved to locally decondense regions to assist in factor binding. The best studied of these complexes is the highly conserved SWI/SNF complex, found in yeast, flies and mammals, that is required for the activation of many, but not all genes. These complexes are very large (~2-MDa) and are composed of 8-11 polypeptides. Recent work with the purified yeast and mammalian complexes has elucidated many of the biochemical properties involved in chromatin remodeling; though many aspects regarding the in vivo biological functions are unclear. For example, while only one subunit has any identified catalytic activity, the remaining subunits are necessary for full in vivo function, modulating the complex activities or targeting it to specific genes or processes. Significant questions remain, such as why so many subunits and how do they individually contribute to the functions of the complex? Do any of the subunits have roles independent of the complex? Also, the complex appears to influence gene regulation both positively and negatively (activation and repression). How is this accomplished and is the regulation direct or indirect? What are the in vivo targets of the complex and how are they selected? Drosophila offers an ideal system for examining these questions, with the full array of genetic, biochemical and cell biological tools available, including a completed genome sequence and a wealth of existing mutations. Drosophila research also provides a detailed developmental framework to bridge these disciplines.This project utilizes molecular, genetic and biochemical analysis of the Drosophila SWI/SNF complex, known as the Brahma (BRM) complex to address these questions. The efforts are focused on one of the most highly conserved and critically important components, known as SNR1. This subunit is crucial in both flies and humans for coordinating or targeting specific protein interactions between the complex and a variety of transcription factors and cell cycle regulatory proteins. A recently isolated temperature sensitive snr1 mutant allows for conditional removal of snr1 function, and the project takes full advantage of this property to fully characterize the biological requirements for SNR1 during development. This is especially important as the snr1 gene is essential in flies and loss of its human counterpart has been strongly correlated with aggressive childhood cancers. In addition to genetic and biochemical studies, DNA microarray analyses using RNA isolated from homozygous mutants at the restrictive temperature will be used to gain a much needed view of the range of targets of the complex in higher eukaryotes, setting the stage for a full investigation of how those targets are selected and regulated in developing tissues. The broader impact of the research is that, through the use of a conditional mutation that is unique among the metazoan SWI/SNF complexes, the biological significance of chromatin remodeling in developmental processes can be better defined by examining in detail when and where the complex is required, and for what functions.

所有多细胞真核生物面临的一个基本挑战是在整个发育过程中协调不同细胞中至关重要的基因的表达，以产生功能齐全的成体。这种协调是通过细胞之间的短程感应信号来完成的，这些信号依赖于可扩散肽和/或直接细胞接触来启动细胞内信号级联，最终影响特定靶基因的表达。染色质已成为细胞转录和复制机制必须应对的主要障碍之一。转录或复制 DNA 所需的因子必须能够进入包装到核小体和更高阶结构中的调控位点。当细胞处于有丝分裂且染色质极度浓缩时，这尤其成问题。能量依赖性染色质重塑复合物已进化为局部解压缩区域以协助因子结合。这些复合物中研究最充分的是高度保守的 SWI/SNF 复合物，存在于酵母、果蝇和哺乳动物中，它是许多但不是所有基因激活所必需的。这些复合物非常大 (~2-MDa)，由 8-11 个多肽组成。最近对纯化酵母和哺乳动物复合物的研究阐明了染色质重塑涉及的许多生化特性。尽管有关体内生物学功能的许多方面尚不清楚。例如，虽然只有一个亚基具有任何已识别的催化活性，但其余亚基对于完整的体内功能、调节复杂的活性或将其靶向特定的基因或过程是必需的。重要的问题仍然存在，例如为什么有这么多的亚基以及它们如何单独贡献复合体的功能？是否有任何亚基具有独立于复合体的作用？此外，该复合物似乎对基因调控有积极和消极的影响（激活和抑制）。这是如何实现的？监管是直接的还是间接的？该复合物的体内靶点是什么以及如何选择它们？果蝇为检查这些问题提供了一个理想的系统，具有全套遗传、生化和细胞生物学工具，包括完整的基因组序列和大量现有突变。果蝇研究还提供了一个详细的发育框架来连接这些学科。该项目利用果蝇 SWI/SNF 复合体（称为 Brahma (BRM) 复合体）的分子、遗传和生化分析来解决这些问题。这些工作的重点是最高度保守且至关重要的成分之一，即 SNR1。该亚基对于果蝇和人类来说对于协调或靶向复合物与各种转录因子和细胞周期调节蛋白之间的特定蛋白质相互作用至关重要。最近分离的温度敏感 snr1 突变体允许有条件地去除 snr1 功能，该项目充分利用这一特性来充分表征开发过程中 SNR1 的生物学要求。这一点尤其重要，因为 snr1 基因在果蝇中至关重要，而人类对应基因的缺失与侵袭性儿童癌症密切相关。除了遗传和生化研究之外，在限制性温度下使用从纯合突变体中分离的 RNA 进行 DNA 微阵列分析将用于获得对高等真核生物中复合物靶标范围的急需了解，为全面研究奠定了基础。如何在发育组织中选择和调节这些目标。该研究更广泛的影响是，通过使用后生动物 SWI/SNF 复合体中独特的条件突变，通过详细检查复合体何时何地出现，可以更好地定义发育过程中染色质重塑的生物学意义。需要什么，以及用于什么功能。