Reconstituting heterochromatin and gene silencing in vivo

体内异染色质重建和基因沉默

• 批准号: 9896664
• 负责人: Andy Yuan
• 金额: $ 1.55万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9896664
• 关键词: Amino Acids; Arginine; Binding; Binding Sites; Biological Assay; Biology; Catalytic Domain; Cells; ChIP-seq; Chromatin; Color; Complex; Coupled; DNA; DNA Polymerase II; DNA-Directed RNA Polymerase; Defect; Deposition; Development; Digestion; Disease Progression; Drug Targeting; Environment; Epigenetic Process; Event; Fission Yeast; Future; Gene Expression; Gene Silencing; Genetic; Genetic Transcription; Genome; Genome Stability; Goals; HDAC4 gene; Heritability; Heterochromatin; High-Throughput Nucleotide Sequencing; Histone Deacetylase; Histone Deacetylation; Histones; Human; Inherited; Length; Liquid Chromatography; Lysine; Maintenance; Malignant Neoplasms; Measures; Mediating; Methylation; Methyltransferase; Micrococcal Nuclease; Modeling; Monitor; Nucleosomes; Organism; Pathway interactions; Phenotype; Play; Polymerase Chain Reaction; Process; Protein Family; Proteins; Proteomics; Quantitative Reverse Transcriptase PCR; Reporter; Reporter Genes; Research; Research Proposals; Role; Saccharomyces cerevisiae; Saccharomycetales; Scheme; System; Testing; Transact; Transcription Coactivator; Work; base; chromatin immunoprecipitation; deep sequencing; dimer; experimental study; genomic locus; high throughput screening; human disease; in vivo; mutant; next generation sequencing; novel therapeutics; promoter; protein complex; protein function; reconstitution; recruit; small molecule; synthetic biology; tandem mass spectrometry; transcription factor; transmission process

PROJECT SUMMARY Heterochromatin plays critical roles in maintaining genome stability and transcriptional gene silencing (TGS). It has become increasingly clear that misregulation of pathways influencing heterochromatin integrity cause or contribute to many human maladies, including numerous cancers. The composition and function of heterochromatin domains are largely conserved from the fission yeast Schizosaccharomyces pombe to humans and other metazoans. Heterochromatin establishment, epigenetic maintenance, and TGS in these organisms are complex processes regulated by numerous chromatin-associated factors. Despite such complexity, core principles of heterochromatin biology have been proposed but remain speculative. This study will experimentally test and articulate these core principles by distilling essential features of heterochromatin in a highly-controlled and orthogonal environment. This research proposal is composed of two aims. Aim 1 is to reconstitute histone 3 lysine 9 methylation (H3K9me)-dependent heterochromatin in Saccharomyces cerevisiae cells, which naturally lack H3K9me, with the goal of defining the minimal requirements for a repressive and heritable chromatin state conserved from fission yeast to human. Successful construction of an H3K9me-dependent heterochromatin domain in S. cerevisiae cells will provide a unique system for investigating how heterochromatin is epigenetically inherited and how it silences transcription and limits other DNA transactions. Accordingly, Aim 2 is to utilize in vivo reconstituted H3K9me-dependent heterochromatin to investigate the mechanism of TGS. H3K9me-dependent heterochromatin will be reconstituted in S. cerevisiae cells by sequentially recruiting S. pombe and human heterochromatin-associated proteins to a specific S. cerevisiae genomic locus. This minimal heterochromatin domain will then be utilized to test three models of TGS and assess the contribution of histone deacetylation and nucleosome remodeling to the formation of silent chromatin domains. A combination of synthetic biology-, next generation sequencing-, and proteomics-based experimental approaches will be utilized to execute this research plan. This work has potential to transform our mechanistic understanding of heterochromatin formation and thereby inform future studies aimed at reversing defects in heterochromatin-associated processes underlying human diseases. Furthermore, the reconstitution of heterologous heterochromatin domains in vivo with factors found in S. pombe and human cells will facilitate the development of cell-based assays amenable to high-throughput screens for small molecules that modulate the function of proteins and protein complexes involved in heterochromatin formation and disease progression. The proposed studies will thus deepen our understanding of fundamental processes underlying human diseases and open new avenues to their treatment.

项目摘要 异染色质在维持基因组稳定性和转录基因沉默（TGS）中起着关键作用。它 越来越清楚的是，影响异染色质完整性的途径的错误调节导致或 导致许多人类疾病，包括许多癌症。的组成和功能 异染色质结构域在很大程度上是从裂殖酵母裂殖酵母保守的， 人类和其他后生动物。异染色质的建立，表观遗传的维持，和TGS在这些 生物体是由许多染色质相关因子调节的复杂过程。尽管有这样 复杂性，异染色质生物学的核心原则已经提出，但仍然是推测性的。本研究 我将通过提取异染色质的基本特征，在实验上测试和阐明这些核心原则， 一个高度受控的正交环境本研究方案由两个目标组成。目标1： 重组酵母中依赖组蛋白3赖氨酸9甲基化（H3K9me）的异染色质 酿酒酵母细胞，其天然缺乏H3K9me，其目标是定义用于H3K9me的最低要求。 从裂殖酵母到人类的抑制性和可遗传的染色质状态。成功建设一个 H3K9me依赖的异染色质结构域。酿酒酵母细胞将提供一个独特的系统， 研究异染色质是如何表观遗传的，以及它是如何沉默转录和限制其他 DNA交易因此，目的2是利用体内重建的H3K9me依赖性异染色质， 探讨TGS的作用机制。H3K9me依赖性异染色质将在S.酿酒酵母 细胞通过顺序募集S.粟酒裂殖酵母和人异染色质相关蛋白的特异性。 酿酒酵母基因座。然后将利用该最小异染色质结构域来测试三种模型， TGS和评估组蛋白去乙酰化和核小体重塑对沉默的形成的贡献。 染色质结构域。基于合成生物学、下一代测序和蛋白质组学的组合 将采用实验方法来执行这项研究计划。这项工作有可能改变我们的 异染色质形成的机制的理解，从而为未来的研究提供信息，旨在逆转 在人类疾病基础的异染色质相关过程中的缺陷。此外，重组 异源异染色质结构域在体内与S.粟酒和人类细胞将促进 开发基于细胞的检测方法，以高通量筛选调节细胞凋亡的小分子， 蛋白质和蛋白质复合物的功能参与异染色质形成和疾病进展。 因此，拟议的研究将加深我们对人类基本过程的理解。 疾病，并开辟新的治疗途径。