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

点击翻译按钮获取中文摘要

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.