RNAi-directed assembly of heterochromatin in Drosophila

果蝇中 RNAi 指导的异染色质组装

基本信息

批准号：
7171917
负责人：
Sarah C.R. ELGIN
金额：
$ 29.74万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-02-01 至 2009-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7171917
关键词：
Antibodies Appearance Aubergine Binding Biochemical Biochemical Genetics Biochemistry Biological Assay Candidate Disease Gene Cell Nucleus Centromere Chromatin Structure Chromosomes Classification Collaborations Collection Complex DNA DNA Methylation DNA Transposable Elements Deacetylation Development Diploidy Double-Stranded RNA Drosophila genome Drosophila genus Drosophila melanogaster Elements Employee Strikes Enzymes Euchromatin Event Failure Figs - dietary Fission Yeast Gene Silencing Gene Targeting Genes Genetic Genome Genomics Goals Health Heterochromatin Histone Deacetylation Histone H3 Histones Homelessness Indium Investigation Knock-out Lead Light Link Localized Lysine Maintenance Malignant Neoplasms Mass Spectrum Analysis Methods Methylation Methyltransferase Missouri Modeling Modification Molecular Biology Mutation Nuclear Structure Nucleosomes Pathway interactions Pattern Phenotype Placement Play Positioning Attribute Principal Investigator Process Proteins Protocols documentation RNA Helicase RNA Interference Range Regulation Reporting Research Design Research Personnel Retroviridae Role SU(VAR)3-9 Series Site Small Interfering RNA Specificity Staining method Stains Standards of Weights and Measures Structure System Techniques Tertiary Protein Structure Testing Thick Thinking Transcript Transgenes Uncertainty Universities Update Work base density design fly heterochromatin-specific nonhistone chromosomal protein HP-1 human DICER1 protein insight knock-down loss of function loss of function mutation member mutant null mutation plant fungi programs protein function research study telomere tissue/cell culture

项目摘要

DESCRIPTION (provided by applicant): Genes normally present in euchromatic domains 0are silenced when packaged into heterochromatin, as exemplified in Drosophila melanogaster by Position Effect Variegation (PEV). Loss-of-function mutations that result in a loss of silencing have identified many critical components of heterochromatin. Heterochromatin formation in pericentric regions appears to be dependent on histone deacetylation, followed by methylation of histone H3 at lysine 9 to generate H3-mK9, and association of Heterochromatin Proteins 1 and 2 (HP1 and HP2). But how is heterochromatin formation targeted to appropriate regions of the genome? We have recently demonstrated that the RNA interference machinery plays a role in Drosophila, as previously reported for S. pombe. We observe that silencing of transgenes embedded in heterochromatin is lost (suppression of PEV) as a result of mutations in piwi, aubergine, or homeless (spindle-E), all of which encode RNAi components. Mutation of homeless leads to a reduction in H3-mK9, and a dramatic delocalization of HP1. We suggest that a "Targeting Complex" containing siRNAs homologous to repetitious elements in heterochromatin can direct specific association of HP1 with these regions. We will test this model by 1) testing candidate loci for loss-of-function mutations that produce the above phenotypes, identifying other RNAi components and histone modification enzymes involved; 2) using a TAP-tag approach to isolate protein complexes associated with three key proteins, HOMELESS, a PAZ/PIWI domain protein (known to bind siRNA), and SDS3, a critical component of the Sin3 histone modification complex, to determine the pattern of interactions; and 3) developing an assay for these phenotypes (loss of H3m-K9, delocalization of HP1) in tissue culture cells to facilitate an unbiased screen for critical factors. The results of these investigations should elucidate the RNAi/heterochromatin pathway, providing new insights into mechanisms of gene silencing, a critical process in development, relevant to many health problems, including cancer.

描述（由申请人提供）：通常存在于构体域中的基因0当包装到异染色质中时沉默，如果蝇中通过位置效应变量（PEV）所示。导致沉默损失的功能丧失突变已经确定了异染色质的许多关键成分。周围区域中的异染色质形成似乎取决于组蛋白脱乙酰基，然后在赖氨酸9处组蛋白H3甲基化以产生H3-MK9，以及异染色质蛋白1和2（HP1和HP2）的缔合。但是，如何针对基因组的适当区域的异染色质形成？我们最近证明，RNA干扰机械在果蝇中起作用，如先前报道的pombe。我们观察到，由于Piwi，Aubergine或无家可归者（Spindle-e）突变，嵌入异染色质中嵌入的转基因的沉默是丢失的（PEV的抑制），所有这些都编码了RNAi成分。无家可归者的突变导致H3-MK9的降低和HP1的戏剧性离域化。我们建议，含有异染色质中重复元素的siRNA同源元素的“靶向复合物”可以将HP1的特定关联与这些区域指导。我们将通过1）测试候选基因座的功能丧失突变，这些突变产生了上述表型，鉴定了其他RNAi成分和涉及的组蛋白修饰酶； 2）使用TAP-TAG方法分离与三种关键蛋白相关的蛋白质复合物，即无家可归者，PAZ/PIWI结构域蛋白（已知可以结合siRNA）和SDS3（SIN3组蛋白修饰复合物的关键成分），以确定相互作用的模式； 3）在组织培养细胞中为这些表型（HP1的损失，HP1的脱位）开发一种测定法，以促进关键因素的无偏筛查。这些研究的结果应阐明RNAi/异染色质途径，从而提供有关基因沉默机制的新见解，基因沉默的机制，这是与包括癌症在内的许多健康问题有关的发展的关键过程。