Live cell imaging of IgH and c-Myc gene loci and the role of nuclear organization

描述（由申请人提供）：该申请涉及广泛的挑战领域（15）转化科学和特定的挑战主题，15- ca -101细胞结构在正常和肿瘤细胞生物学中的作用。细胞核是高度有序的细胞器。基因位点和蛋白质动态定位于细胞核内的区室，区室化被认为调节转录和促进核生理。然而，也可能对该组织产生负面影响。在受刺激的B细胞中，IgH和c-Myc位点经常共定位于共享的转录工厂（浓缩活性RNA聚合酶II的位点），并且在浆细胞瘤和伯基特淋巴瘤中，IgH和c-Myc是频繁的易位伙伴。这种相关性表明，这两个基因在转录工厂内的物理接近导致了染色体易位的高频率。为了确定基因最初与转录工厂相关的机制，并验证基因座的共定位是产生IgH/c-Myc易位的致病因素的假设，将使用4D（三维随时间）荧光显微镜对活细胞中的IgH和c-Myc基因座进行成像。细胞核结构和功能研究领域的一个主要挑战是揭示细胞核组织建立的机制及其在生理上的作用。造成这一挑战的原因之一是缺乏对活细胞的实时分析，以及在自然发育和疾病状态下的实时分析。相反，在细胞核中建立秩序的机制以及核组织对正常和疾病生理的贡献都是从静态数据点推断出来的，因此导致了对数据的多种解释和未解决的假设。在这里，我们将使用4D荧光显微镜来解决这一挑战。具体来说，我们将测试相互排斥的假设：(1)基因移动到预先存在的转录工厂，包括在前b细胞到前b细胞成熟期间的IgH，以及在CD40/IL-4刺激期间c-Myc移动到由IgH预先占据的转录工厂；(2)转录工厂在基因上重新成核。4D显微镜将使我们能够在同一实验中通过直接可视化来区分这些模型。此外，IgH和c-Myc位点之间的距离将通过淋巴生成来测量，并用于确定B细胞刺激前两个位点之间的距离是否会影响它们共定位到同一转录工厂的概率。然后将c-Myc基因座连接到核孔复合物上，将c-Myc基因座与IgH基因座强行分离，并确定该核室是否允许转录。最后，我们将确定强行分离IgH和c-Myc基因座是否会降低IgH/c-Myc易位的频率和/或增加IgH易位到其他基因座的频率。我们的总体策略将是创建一个小鼠胚胎干细胞系，从一个多顺吞盒中表达荧光标记的蛋白质。荧光蛋白融合物将标记关键的核室和特定的位点（通过荧光标记LacI和TetR结合到LacO和TetO结合位点阵列）。LacO和TetO阵列将分别集成在c-Myc和IgH位点上，以标记这些基因。然后将修饰的胚胎干细胞分化为b细胞，并使用荧光显微镜成像以验证上述假设。值得注意的是，这个项目的设计是在不同的实验条件下使用单个细胞系，从而提高了数据生成的速度和效率。虽然我们将在这里提出的工作中测试重要的机制假设，但还有许多其他问题超出了这个为期两年的提案的范围，可以使用这些相同的工具来解决。因此，本项目将为今后的长期研究奠定基础。最后，由于刺激美国经济是这项区域投资基金的一个目标，我们将创造多个就业机会，购买几件设备，支持弗雷德哈钦森癌症中心的核心服务，并雇用外部生物技术公司的服务。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (15) Translational Science and specific Challenge Topic, 15-CA-101 The Role of Cellular Architecture in Normal and Tumor Cell Biology. The cell nucleus is a highly ordered organelle. Gene loci and proteins are dynamically localized to compartments within the nucleus, and compartmentalization is thought to regulate transcription and promote nuclear physiology. However, there also may be negative consequences to this organization. In stimulated B cells, the IgH and c-Myc loci often colocalize in shared transcription factories (sites of concentrated active RNA polymerase II), and IgH and c-Myc are frequent translocation partners in plasmacytomas and Burkitt lymphoma. This correlation suggests that the physical proximity of these two genes within transcription factories leads to the high frequency of chromosomal translocations. To determine the mechanism by which genes initially associate with transcription factories and to test the hypothesis that colocalization of the loci is a causative factor in generating IgH/c-Myc translocations, the IgH and c-Myc loci will be imaged in live cells using 4D (3 dimensions over time) fluorescence microscopy. A major challenge in the field of nuclear structure and function has been to uncover the mechanisms underlying the establishment of nuclear organization and to discover its role in physiology. Contributing to the challenge has been a lack of real time assays in live cells, and in the context of native states of development and disease. Instead, the mechanisms underlying the establishment of order in the nucleus and the contribution of nuclear organization to normal and disease physiology have been inferred from static data points thus leading to multiple interpretations of the data and unresolved hypotheses. Here, we will use 4D fluorescence microscopy to address this challenge. Specifically, we will test the mutually exclusive hypotheses that (1) genes move to pre-existing transcription factories, including IgH during pro-B-cell to pre-B cell maturation and c-Myc to transcription factories pre-occupied by IgH during ¿CD40/IL-4 stimulation and (2) that transcription factories nucleate de novo on genes. 4D microscopy will enable us to distinguish these models by direct visualization in the same experiment. Further, the distance between the IgH and c-Myc loci will be measured through lymphopoiesis and used to determine if the distance between the two loci prior to B cell stimulation influences the probability of their colocalization to the same transcription factory. The c-Myc locus will then be tethered to nuclear pore complexes to forcibly separate the c-Myc locus from the IgH locus and to determine if this nuclear compartment is permissible to transcription. Finally we will determine if forced separation of the IgH and c-Myc loci decreases the frequency of IgH/c-Myc translocations and/or increases the frequency of IgH translocations to other loci. Our general strategy will be to create a line of murine ES cells expressing fluorescently tagged proteins from a polycistonic cassette. The fluorescent protein fusions will label key nuclear compartments and specific loci (via fluorescently tagged LacI and TetR binding to arrays of LacO and TetO binding sites). LacO and TetO arrays will be integrated at the c-Myc and IgH loci, respectively to label those genes. The modified ES cells will then be differentiated into B-cells and imaged using fluorescence microscopy to test the above hypotheses. Notably, this project has been designed so that a single cell line will be used under different experimental conditions, thereby increasing the speed and efficiency of data production. While we will test significant mechanistic hypotheses in the work proposed here, there are many additional questions beyond the scope of this 2-year proposal that can be addressed using these same tools. Thus this project will provide a foundation for long-term studies. Finally, as stimulating the American economy is an objective for this RFA, we will create multiple jobs, purchase several pieces of equipment, support core services at the Fred Hutchinson Cancer Center, and employ the services of external biotechnology companies. PUBLIC HEALTH RELEVANCE: The relationship between the position of gene loci in the nucleus and cell physiology has emerged as an important facet of cell biology. Chromosomal translocations occur when double stranded breaks in DNA are repaired by incorrectly fusing one end of the break to a break in another chromosome, often leading to malignant transformation. It has been proposed that colocalization of gene loci into shared compartments predisposes them to translocations. Here we will test those predictions in the context of Burkitt Lymphoma using the common translocation partners IgH and c-Myc. These two gene loci also frequently colocalize in so- called transcription factories. We will first determine the mechanism by which these gene loci interact with transcription factories and then determine if forcibly separating the two loci decreases the frequency of their translocations.