Genes and chromosomes are non-randomly positioned within the nucleus, meaning they tend to be preferentially positioned either in relation to the center/edge of the nucleus or relative to other genes and chromosomes. For example in the microscopy image of developmentally immature red blood cell nuclei on the right, both copies of the beta-globin gene (labeled in green by a technique called DNA FISH) are adjacent to the nuclear lamina which surrounds the inside surface of the nucleus and is labeled red. The blue color is a dye that labels all DNA. There is also a link between this so called nuclear organization and both development and disease. For example, certain blood cancers are caused by double strand breaks in the DNA being incorrectly repaired such that the ends are fused to another chromosome instead of each other. These chromosome fusions are called translocations. In solid tumors, translocations are also associated with disease progression. Many of these translocations are recurrent, meaning the same breakpoints are always involved and it turns out those breakpoints are frequently physically close to one another in the nucleus, even in cells without the double strand break.
In a recently published study, we investigated the mechanism controlling the close positioning of two genes involved in a recurrent chromosomal translocation that causes Burkitt’s lymphoma in humans and a similar cancer called plasmacytoma in mice. We found that these two genes, IgH and Myc, are positioned close to each other because the chromosomes containing those genes are both tethered to the nucleolus. The nucleolus is an organelle inside the nucleus that has several functions, the most prominant is synthesizing the components of ribosomes. See the image of a mouse B-cell nucleus to the right. Here we labeled both copies of Chromosome 12 teal and both copies of Chromosome 15 yellow using a technique called Whole Chromome Painting. The nucleolus was labeled orange using immunofluorescence. You can see a few things in this image. First, each chromosome occupies a limited volume rather than being spread out all over the nucleus. These are called chromosome territories. Note that it is not the X shaped thing you often see diagrammed for chromosomes. That is what chromosomes look like when they are dividing but these cells are in interphase, i.e. they are just hanging out doing their thing. The second point, which is most relevant to this research is that both copies of the two different chromosomes are touching nucleoli and they are close to each other. The scale bar is 1 micron = 1/1000 of a mm.
When we look at the IgH and Myc genes themselves we see a similar organization. In the image to the right we see a different mouse B-cell. In this case both copies of the IgH gene were labeled teal and both copies of the Myc gene were labeled yellow using DNA FISH. The nucleolus was again labeled orange by immunofluorescence and the grey color is the dye that labels all DNA. You can see that both IgH and Myc are adjacent to the nucleolus and adjacent to each other. Note that this cell has 1 nucleolus and the previous cell had two. There is cell to cell variability in the number of nucleoli, but the average is about 2 1/2 per cell. The scale bar is 1 micron.
Even though IgH and Myc are physically close to one another, they are very rarely in direct physical contact. In order for a tranlocation to occur however, the ends of a double strand break need to directly fuse to a break on the other chromosome. This implies then, that the IgH and Myc genes move before contact each other when forming a translocation. Data from other labs support this model and it is important to note that the movement is diffusive, not directed. There is also pretty strong evidence in the literature that the probability of a translocation is increased when one of the molecular pathways responsible for DNA break repair, fails. When that happens, the break persists for longer, meaning there is a greater chance for bad stuff to happen and alternate break repair pathways are activated. The alternate pathways are more likely to permit a translocation than the primary repair pathways.
My current research focuses on these dynamic aspects of DNA break repair, that is, the competition between different break repair pathways and the movement of DNA breaks within the nucleus. I am using breaks in both native genes and introduced synthetic constructs along with genetic manipulations to look at the spatial and temporal regulation of DNA repair, using mammalian tissue culture cells as a model system. I am also investigating how the local conditions at the break site influence repair pathway choice and ultimately the outcome of the repair. The goal is to better understand the series of events that occurs upon DNA damage in the context of the nucleus and by better understanding the fundamental processes we can better understand how defects in these processes lead to disease.