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.
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.
| Strongin, Daniel E; Groudine, Mark; Politz, Joan C Ritland (2014) Nucleolar tethering mediates pairing between the IgH and Myc loci. Nucleus 5:474-81 |
