Investigating the Role of Histone Modifications in Heterochromatin Formation

研究组蛋白修饰在异染色质形成中的作用

基本信息

批准号：
9114871
负责人：
Taylor Joel Richard Penke
金额：
$ 3.13万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-01 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9114871
关键词：
Acetylation Address Affect Amino Acids Anaphase Animals Biological Assay Biological Process Biology Cell physiology Centromere ChIP-seq Charge Chromatin Chromatin Structure Chromosomal Breaks Chromosome Segregation Chromosome Structures Chromosomes Cognition Disorders Confocal Microscopy Cytology DNA DNA Damage DNA Transposable Elements Data Set Defect Disease Disease Progression Drosophila genus Engineering Environment Enzymes Eukaryota FAIRE sequencing Gene Expression Genes Genetic Recombination Genomic Segment Genomics Heterochromatin Histone H3 Histones Huntington Disease Immunofluorescence Immunologic Investigation Knowledge Larva Lead Location Lysine Malignant Neoplasms Mediating Methylation Methyltransferase Mitotic Chromosome Modeling Modification Mutate Mutation Physical condensation Positioning Attribute Post-Translational Protein Processing Process Proteins RNA Recruitment Activity Regulation Repression Role Salivary Glands Side Structure Testing Therapeutic Intervention Time Wing anti-cancer therapeutic chromatin protein genome-wide genome-wide analysis heterochromatin-specific nonhistone chromosomal protein HP-1 histone modification mutant neuroblast non-histone protein novel prevent public health relevance

项目摘要

DESCRIPTION (provided by applicant): Heterochromatin is implicated in the regulation of diverse biological processes including recombination, chromosome segregation, transposon repression, and gene expression. As such, mutations in heterochromatin associated proteins are recurrently found in a variety of diseases, including Huntington's disease and numerous cancers. However, the role of these mutations in the progression of disease is poorly understood. A deeper understanding of heterochromatin regulation and the consequences of altered heterochromatin environments will be essential for understanding potential connections to disease. One potential mechanism of heterochromatin regulation is post-translational modification (PTM) of histones, including methylation of lysine nine on histone H3 (H3K9me). Several labs have attempted to study the role of H3K9me by mutating the enzymes that establish this modification. However, conclusions from their studies are limited because these enzymes have several non-histone substrates in addition to H3K9. Unfortunately, directly testing the role of H3K9 by mutating this residue to a non-modifiable amino acid has been unfeasible in higher eukaryotes due to the difficulty of engineering replacement histone genes. For this reason, although modification of H3K9 has been considered an important regulator of heterochromatin for more than a decade, the function of H3K9 in animals has never been directly tested. By using a Drosophila histone replacement platform recently established by our lab and collaborators, we will analyze the contribution of H3K9 to heterochromatin regulation by replacing endogenous histones with H3K9 mutant histones. The first aim of this study seeks to determine if mutating H3K9 results in altered heterochromatin structure by assessing recruitment of heterochromatin associated proteins and chromatin compaction. Given the ability of H3K9me to recruit heterochromatin factors we expect some genomic locations to lose heterochromatic proteins and consequently form a more open chromatin environment. We will test this idea by examining localization of Heterochromatin Protein 1 and heterochromatin associated histone PTMs via ChIP-seq and polytene chromosome cytology. Moreover, we will genetically assess heterochromatin formation using Position-Effect Variegation assays and explore genome-wide open chromatin profiles using FAIRE-seq to interrogate chromatin structure in H3K9 mutants. The second aim of this proposal will address the functional importance of H3K9 for two heterochromatin regulated processes, transposon repression and chromosome segregation. We hypothesize that a compromised heterochromatic environment will lead to de-repression of transposable elements and defects in chromosome segregation. We will test these hypotheses by performing quantitative PCR to analyze transposon expression and confocal microscopy to investigate chromosome segregation. Ultimately, we expect analysis of H3K9's role in regulating heterochromatin to augment our understanding of how altered chromatin structure may lead to disease.

描述（适用提供）：异染色质与潜水生物学过程的调节有关，包括重组，染色体分离，转座子表达和基因表达。因此，异染色质相关蛋白的突变在包括亨廷顿氏病和众多癌症在内的多种疾病中经常发现。但是，这些突变在疾病进展中的作用知之甚少。对异染色质调节的更深入了解以及改变异染色质环境的后果对于理解潜在的与疾病的联系至关重要。异染色质调节的一种潜在机制是组蛋白的翻译后修饰（PTM），包括组蛋白H3（H3K9ME）上赖氨酸九的甲基化。一些实验室试图通过突变建立这种修饰的酶来研究H3K9ME的作用。但是，他们的研究得出的结论是有限的，因为除了H3K9之外，这些酶还具有几种非固件底物。不幸的是，由于工程替换组蛋白基因的困难，在较高的真核生物中直接测试H3K9的作用是在较高的真核生物中不可行的。因此，尽管H3K9的修饰已被认为是杂染色质的重要调节剂，但是十多年来，H3K9在动物中的功能从未直接测试。通过使用我们的实验室和合作者最近建立的果蝇组蛋白替代平台，我们将通过用H3K9突变体组蛋白代替内源性组蛋白来分析H3K9对异染色质调节的贡献。这项研究的第一个目的旨在确定通过评估异染色质相关蛋白和染色质压实的募集来确定H3K9是否会导致异染色质结构改变。鉴于H3K9ME募集异染色质因子的能力，我们期望某些基因组位置失去异质蛋白，从而形成更开放的染色质环境。我们将通过检查异染色质蛋白1和异染色质组蛋白PTM的定位来测试这一想法，并通过chip-seq和多丁烯染色体细胞学研究。此外，我们将使用位置效应变化测定法评估杂染色质形成，并使用Faire-Seq探索全基因组开放式染色质特征，以询问H3K9突变体中的染色质结构。该提案的第二个目的将探讨H3K9对于两个异染色质的过程，转座子反射和染色体隔离的功能重要性。我们假设造成的异质环境将导致染色体隔离中的转座元素和缺陷的抑制。我们将通过执行定量PCR来分析转座子表达和共聚焦显微镜以研究染色体分离来检验这些假设。最终，我们期望分析H3K9在调节异染色质中的作用，以增强我们对染色质结构如何导致疾病的理解。