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

染色体在细胞核内以复杂的方式组织起来。例如，单个基因组基因座 通过循环、配对和亚核靶向，呈现出不同的构象。绝缘子和聚合体 反应元件(PreS)，绝缘体蛋白结合的DNA元件和多梳基复合体， 调停这些互动。核的不适当组织与包括乳房在内的疾病有关 癌症、胰腺癌和肢体畸形，但尚不清楚核靶向和远距离 特定基因组位点之间的相互作用有助于维持适当的基因表达。 核组织在随机基因表达中起着至关重要的作用，它在基因表达过程中被使用 使细胞命运多样化的发展。随机基因表达的中断可导致自闭症、视力和 嗅觉障碍、淋巴瘤和免疫缺陷，但对其控制机制知之甚少 基因的随机开/关表达。这个项目的目标是确定亚核如何 区隔和远距离基因相互作用控制着随机表达的决定。 研究随机基因调控的一个很好的模型是果蝇视网膜，在那里基因没有刺 (SS)在光感受器细胞子集中以随机开/关的方式表达。两种机制控制 随机SS表达：1)“表达决定”，即核内SS的每个拷贝都产生一个 自主决定开启或关闭，以及2)“染色体间通讯(内部通讯)”，其中 单个ss拷贝之间的串扰协调ss表达频率。党卫军核定位变化 SS-ON和SS-OFF细胞类型之间的差异，表明SS亚核位置对表达至关重要 决定。此外，SS的拷贝在细胞核内配对，与它们在基因组中的位置无关， 这表明对讲机要求党卫军的副本必须在物理上非常接近。此外，具体来说， SS中的绝缘体和PreS似乎在表达决定、配对和内部通话中起中介作用。我们假设 顺式调控元件指导ss亚核靶向以控制ss表达决定(目标1)和 调解SS配对以控制内部通信(目标2)。我们将进一步研究控制SS的机制。 利用DNA寡核苷酸FISH技术追踪野生型ss和 CRISPR产生的绝缘体和突变前的ss等位基因与激活和抑制核体有关 (目标1)。我们将通过测试对讲能力的配对来进一步研究对讲的机制 利用DNA寡核苷酸对SS转基因和突变等位基因的研究。然后我们将确定哪些DNA元素是 通过检查单个SS的CRISPR和BAC转基因缺失进行配对和内部通信所需的 绝缘体和预制体(目标2)。这个项目的结果将阐明DNA元素之间的相互作用 跨越长距离的核，有助于在发育过程中进行适当的基因调控，并防止疾病状态。

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.