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

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体。这种协调是通过细胞间的短距离感应信号来完成的，这些信号依赖于扩散肽和/或直接的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克服的主要障碍之一。转录或复制DNA所需的因子必须进入被包装成核小体和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。能量依赖的染色质重塑复合体已经进化到局部去密集区域，以协助因子结合。这些复合物中研究得最好的是高度保守的SWI/SNF复合物，在酵母、苍蝇和哺乳动物中发现，它是激活许多基因所必需的，但不是所有基因。这些复合物非常大（~2-MDa），由8-11多肽组成。最近对纯化酵母和哺乳动物复合物的研究已经阐明了染色质重塑中涉及的许多生化特性；虽然在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确定的催化活性，但其余亚基对于充分的体内功能是必需的，可以调节复杂的活性或将其靶向特定的基因或过程。重要的问题仍然存在，比如为什么有这么多亚基，它们是如何单独对复合体的功能做出贡献的？有哪个亚基的作用独立于复合体吗？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，规定是直接的还是间接的？复合物的体内靶点是什么？它们是如何被选择的？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、生化和细胞生物学工具，包括完整的基因组序列和丰富的现有突变。果蝇研究也为这些学科提供了一个详细的发展框架。该项目利用果蝇SWI/SNF复合体（即Brahma （BRM）复合体）的分子、遗传和生化分析来解决这些问题。这些努力集中在一个高度保守和至关重要的组成部分，被称为SNR1。该亚基在果蝇和人类中都是至关重要的，用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。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snr1在发育过程中的生物学需求。这一点尤其重要，因为snr1基因在果蝇中是必不可少的，而人类snr1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，将用于获得高等真核生物中复合体靶点范围的急需视图，为全面研究这些靶点在发育组织中是如何选择和调节的奠定基础。该研究的更广泛的影响是，通过使用后生动物SWI/SNF复合体中独特的条件突变，可以通过详细检查何时何地需要复合体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。

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.