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

在所有多细胞真核生物中，一个根本的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之间的短程诱导信号来完成的，这些信号依赖于可扩散的多肽和/或直接的细胞接触来启动细胞内的信号级联，最终影响特定靶基因的表达。染色质已经成为细胞转录和复制机制必须与之抗衡的主要障碍之一。转录或复制DNA所必需的因子必须能够接触到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，染色质极度浓缩时，这尤其是个问题。依赖于能量的染色质重塑复合体已经进化到局部去致密区域，以帮助因子结合。这些复合体中研究得最好的是高度保守的SWI/SNF复合体，发现于酵母、苍蝇和哺乳动物中，它是激活许多但不是所有基因所必需的。这些复合体非常大(~2-丙二醛)，由8-11个多肽组成。最近对纯化的酵母和哺乳动物复合体的研究已经阐明了与染色质重塑有关的许多生化性质；尽管关于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，但其余的亚基是体内充分发挥功能所必需的，调节复杂的活性或将其靶向于特定的基因或过程。重要的问题仍然存在，例如为什么有这么多的亚单位，它们如何单独对复合体的功能做出贡献？是否有任何亚单位具有独立于复合体之外的角色？此外，该复合体似乎对基因调控(激活和抑制)都有积极和消极的影响。这是如何实现的？监管是直接的还是间接的？该复合体的体内靶点是什么？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，拥有一整套可用的遗传、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的突变。果蝇的研究也提供了一个详细的发展框架来弥合这些规律。该项目利用果蝇SWI/SNF复合体的分子、遗传和生化分析，即Brahma(BRM)复合体来解决这些问题。这些努力的重点是最保守、最关键的组成部分之一，即SNR1。在果蝇和人类中，这个亚基对于协调或靶向该复合体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是至关重要的。最近分离的一个温度敏感的SNr1突变体允许有条件地移除SNr1功能，该项目充分利用这一特性来充分表征发育过程中对SNR1的生物学要求。这一点尤其重要，因为Snr1基因在果蝇中是必不可少的，而其人类对应基因的缺失与儿童侵袭性癌症密切相关。除了遗传和生化研究外，还将使用在限制性温度下从纯合子突变体中分离出的RNA进行DNA微阵列分析，以获得高等真核生物中该复合体靶标范围的迫切需要的观点，为全面研究这些靶标在发育中的组织中如何选择和调节奠定基础。这项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，可以通过详细研究何时何地需要该复合体以及用于什么功能来更好地确定染色质重塑在发育过程中的生物学意义。

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.