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，以产生一个功能齐全的成年人。这种协调是通过细胞之间的短程诱导信号来实现的，这些信号依赖于可扩散的肽和/或直接细胞接触来启动最终影响特定靶基因表达的细胞内信号级联。染色质已经成为细胞转录和复制机制必须克服的主要障碍之一。转录或复制DNA所需的因子必须能够进入包装成核小体和更高级结构的调控位点。当细胞处于有丝分裂和染色质极度浓缩时，这尤其成问题。能量依赖性染色质重塑复合物已经进化到局部去致密区域以帮助因子结合。这些复合物中研究得最好的是在酵母、苍蝇和哺乳动物中发现的高度保守的SWI/SNF复合物，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。最近的工作与纯化的酵母和哺乳动物的复合物已经阐明了许多涉及染色质重塑的生化特性，虽然在体内生物学功能的许多方面还不清楚。例如，虽然只有一个亚基具有任何鉴定的催化活性，但其余的亚基是完整的体内功能所必需的，调节复合物活性或将其靶向特定的基因或过程。重要的问题仍然存在，例如为什么有这么多的亚单位，以及它们如何单独对复合体的功能做出贡献？是否有任何子单位的角色独立于综合体？此外，复合物似乎对基因调控产生积极和消极的影响（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？它们是如何选择的？果蝇为研究这些问题提供了一个理想的系统，拥有完整的遗传、生物化学和细胞生物学工具，包括完整的基因组序列和大量现有的突变。果蝇研究也提供了一个详细的发展框架，以弥合这些学科。本项目利用分子，遗传和生物化学分析果蝇SWI/SNF复合体，称为梵天（BRM）复合体，以解决这些问题。这些努力的重点是最高度保守和至关重要的组成部分之一，称为SNR 1。该亚基在果蝇和人类中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异性蛋白质相互作用至关重要。最近分离的温度敏感的snr 1突变体允许有条件地去除snr 1功能，该项目充分利用了这一特性，以充分表征开发过程中对snr 1的生物学要求。这一点尤其重要，因为snr 1基因在果蝇中是必不可少的，而人类snr 1基因的缺失与侵袭性儿童癌症密切相关。除了遗传和生物化学研究，DNA微阵列分析，使用RNA分离的纯合突变体在限制性的温度将被用来获得一个非常需要的视图的范围内的复杂的目标在高等真核生物，设置阶段的全面调查，这些目标是如何选择和调节在发展中的组织。该研究的更广泛影响是，通过使用后生动物SWI/SNF复合物中独特的条件突变，可以通过详细检查何时何地需要复合物以及功能来更好地定义发育过程中染色质重塑的生物学意义。