Chromatin Organization and RNA silencing

染色质组织和 RNA 沉默

• 批准号: 7733962
• 负责人: Elissa Lei
• 金额: $ 56.83万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733962
• 关键词: 3' -nuclease; Affect; Binding; Biochemical; Breast; Cataloging; Catalogs; Cells; Chromatin; Chromatin Loop; Colorectal Cancer; Complex; DNA; DNA Sequence; Depth; Development; Disruption; Distant; Drosophila genus; Erythroid; Gene Expression; Gene Expression Regulation; Genes; Genome; Gypsies; Higher Order Chromatin Structure; Human; In Situ Hybridization; Lead; Localized; MYC gene; Mutation; Nuclear; Nuclear Matrix; Pancreatic ribonuclease; Pathway interactions; Process; Protein Overexpression; Proteins; RNA Interference; Regulation; Research; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleases; Role; Structure; Techniques; Transcriptional Regulation; Work; beta Globin; in vivo; nuclease; programs; research study

The focus of our work is the gypsy chromatin insulator of Drosophila, the best studied chromatin insulator to date. The gypsy insulator is comprised of a DNA sequence bound by a complex of at least three proteins. Several lines of evidence suggest that insulator proteins bridge distant DNA sequences dispersed throughout the genome, causing looping of the DNA and the creation of a distinct chromatin domain. Nuclear aggregates of insulator complexes termed insulator bodies are tethered stably to the nuclear matrix and may form higher order structures of chromatin loops. Interestingly, insulator body association with the nuclear scaffold can be disrupted by RNase A treatment. These findings prompted us to examine whether RNA silencing, an RNA-dependent cellular mechanism of gene regulation known to act on the level of chromatin, affects gypsy insulator activity. Our research provides evidence for a previously unknown role for RNA silencing in gypsy insulator function as well as higher order chromatin organization. Using biochemical purification techniques, we have identified an RNA-dependent physical interaction between proteins required for proper gypsy insulator and RNA silencing function. Furthermore, mutations in genes encoding RNA silencing components affect gypsy insulator activity in vivo and the formation of insulator bodies. These results suggest that RNAs involved in the RNA silencing pathway are responsible for the multimerization of insulator complexes and/or the ability of insulator bodies to interact with a nuclear scaffold. Our current efforts center on identifying RNAs associated with the gypsy insulator by immunoaffinity purification and cataloguing them by deep sequencing using the Illumina Genome Analyzer platform. Several RNAs identified thus far have been confirmed by RT-PCR and are currently being localized in cells by in situ hybridization. We have also discovered the physical association of several of nucleases that may be involved in the processing of these copurifying RNAs. Experiments are under way to determine if chromatin insulator complexes harbor nuclease activity and whether this enzymatic activity influences insulator function.

我们工作的重点是果蝇的吉普赛染色质绝缘子，迄今为止研究的染色质绝缘子最好。 吉普赛绝缘子由由至少三个蛋白质的复合物结合的DNA序列组成。 几条证据表明，绝缘蛋白桥桥远处的DNA序列分散在整个基因组中，导致DNA循环并产生独特的染色质结构域。 被称为绝缘体体的绝缘体复合物的核骨料稳定地束缚在核基质上，并可能形成染色质环的高阶结构。 有趣的是，RNase A治疗可以破坏绝缘体的体型与核支架的关联。 这些发现促使我们检查了RNA沉默是已知的基因调节的RNA依赖性细胞机制是否影响染色质水平，会影响吉普赛绝缘体活性。 我们的研究提供了以前未知的RNA沉默在吉普赛绝缘子功能以及高阶染色质组织中的作用的证据。 使用生化纯化技术，我们确定了适当的吉普赛绝缘子所需的蛋白质与RNA沉默功能之间的RNA依赖性物理相互作用。 此外，编码RNA沉默成分的基因突变会影响体内吉普赛绝缘子活性和绝缘体的形成。 这些结果表明，参与RNA沉默途径的RNA是导致绝缘子复合物的多聚化和/或绝缘体与核支架相互作用的能力。 我们目前的努力集中在通过使用Illumina Genome Analyzer平台进行深层测序来确定与吉普赛绝缘子相关的RNA与吉普赛绝缘子相关的RNA。 迄今为止，RT-PCR已确认了一些RNA，目前正在通过原位杂交定位于细胞中。 我们还发现了可能参与这些共纯净RNA的几个核酸酶的物理关联。 正在进行实验，以确定染色质绝缘子是否含有核酸酶活性以及该酶活性是否影响绝缘体功能。