Determining the role of 3D nuclear architecture in stochastic gene expression

确定 3D 核结构在随机基因表达中的作用

• 批准号: 9190511
• 负责人: Kayla Chelsea Viets
• 金额: $ 4.36万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-01 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9190511
• 关键词: Alleles; Architecture; Autistic Disorder; B-Lymphocytes; Beryllium; Binding; Cell Nucleolus; Cell Nucleus; Cells; Centromere; Chromosome Mapping; Chromosome Territory; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Color; Communication; Complex; DNA; DNA Sequence; Data; Development; Disease; Drosophila genus; Drosophila melanogaster; Elements; Enhancers; Exposure to; Fluorescent in Situ Hybridization; Frequencies; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Order; Genes; Genetic Transcription; Genome; Genomics; Goals; Immunologic Deficiency Syndromes; Individual; Interneurons; Lead; Limb structure; Link; Location; Lymphoma; Malignant neoplasm of pancreas; Mammals; Mediating; Molecular Conformation; Motor Neurons; Nuclear; Nuclear Lamina; Nucleoplasm; Organism; Photoreceptors; Play; Polycomb; Positioning Attribute; Process; Proteins; Regulatory Element; Response Elements; Retina; Role; Saccharomyces cerevisiae; Specific qualifier value; Study models; Testing; Transgenes; Vision Disorders; Work; base; cell type; chromosomal location; gene interaction; malformation; malignant breast neoplasm; mutant; olfactory disorder; olfactory receptor; prevent; spindle pole body; stem cells; telomere

Chromosomes are organized in a complex manner within the nucleus. For example, individual genomic loci take on distinct conformations via looping, pairing, and subnuclear targeting. Insulators and polycomb response elements (PREs), DNA elements bound by insulator proteins and the Polycomb Group complex, mediate these interactions. Improper organization of the nucleus has been linked to disorders including breast cancer, pancreatic cancer, and limb malformations, but it is unclear how nuclear targeting and long-distance interactions between specific genomic loci work to maintain proper gene expression. Nuclear organization plays an essential role in stochastic gene expression, which is used during development to diversify cell fates. Disruptions in stochastic gene expression can lead to autism, visual and olfactory disorders, lymphoma, and immunodeficiencies, but little is known about the mechanisms that control the random on/off expression of genes. The goal of this project is to determine how subnuclear compartmentalization and long-distance gene interactions control stochastic expression decisions. An excellent model for studying stochastic gene regulation is the fruit fly retina, where the gene spineless (ss) is expressed in a random on/off manner in a subset of photoreceptor cells. Two mechanisms control stochastic ss expression: 1) the “expression decision,” in which each copy of ss within a nucleus makes an independent decision to be either on or off, and 2) “Interchromosomal Communication (InterCom),” in which crosstalk between individual ss copies coordinates ss expression frequency. ss nuclear localization changes between Ss-on and Ss-off cell types, suggesting that ss subnuclear position is critical for the expression decision. Additionally, copies of ss pair within the nucleus independent of their location in the genome, suggesting that InterCom requires copies of ss to be in close physical proximity. Furthermore, specific insulators and PREs within ss appear to mediate expression decisions, pairing, and InterCom. We hypothesize that cis-regulatory elements direct ss subnuclear targeting to control the ss expression decision (Aim 1) and mediate ss pairing to control InterCom (Aim 2). We will further investigate the mechanisms controlling the ss expression decision by using the DNA Oligopaints FISH technique to track the localization of wild-type ss and CRISPR-generated insulator- and PRE-mutant ss alleles relative to activating and repressing nuclear bodies (Aim 1). We will further investigate the mechanisms of InterCom by testing the pairing of InterCom-competent ss transgenes and mutant alleles using DNA Oligopaints. We will then determine which DNA elements are required for pairing and InterCom by examining CRISPR and BAC transgene deletions of individual ss insulators and PREs (Aim 2). The results of this project will elucidate how interactions between DNA elements across long nuclear distances facilitate proper gene regulation during development and prevent disease states.

染色体在细胞核内以复杂的方式组织。例如，单个基因组基因局 通过循环，配对和亚核靶向进行独特的构象。绝缘子和polycomb 响应元件（PRES），由绝缘体蛋白绑定的DNA元素和Polycomb组复合物， 调解这些相互作用。核组织不当的组织与包括乳房在内的疾病有关 癌症，胰腺癌和肢体畸形，但目前尚不清楚核靶向和长距离如何 特定的基因组基因座工作以保持适当的基因表达之间的相互作用。 核组织在随机基因表达中起着至关重要的作用，在此期间使用 发展以使细胞命运多样化。随机基因表达的破坏会导致自闭症，视觉和 嗅觉疾病，淋巴瘤和免疫缺陷，但对控制的机制知之甚少 基因的随机开/关表达。该项目的目的是确定如何亚核 分隔和长距离基因相互作用控制随机表达决策。 研究随机基因调节的一个极好模型是果蝇，其中基因无脊椎 （SS）在感光细胞的子集中以随机的/关节方式表达。两种机制控制 随机SS表达式：1）“表达式决策”，其中核中的每个SS副本都使 独立的决定要么打开或关闭，还有2）“染色体通信（对讲）”，其中 单个SS之间的串扰副本副本协调SS表达频率。 SS核定位变化 在SS-ON和SS-OFF细胞类型之间，表明SS亚核位置对于表达至关重要 决定。此外，在原子核内的SS对的副本独立于基因组中的位置， 暗示对讲需要SS的副本密切的物理接近。此外，具体 SS中的绝缘体和PRES似乎介导了表达决策，配对和对讲。我们假设 顺式调节元件指导SS亚核靶向控制SS表达决策（AIM 1）和 介导SS配对以控制对讲（AIM 2）。我们将进一步研究控制SS的机制 通过使用DNA寡聚鱼技术来跟踪野生型SS的定位和 相对于激活和反映核体，CRISPR生成的绝缘子和突变的SS等位基因 （目标1）。我们将通过测试竞争力的配对来进一步研究对讲机的机制 SS转基因和突变等位基因使用DNA寡聚。然后，我们将确定哪些DNA元素是 通过检查CRISPR和BAC转换单个SS的缺失，需要配对和对讲 绝缘子和PRES（AIM 2）。该项目的结果将阐明DNA元素之间的相互作用 在长时间的核距离上，准备在发育过程中制备了适当的基因调节并预防疾病状态。