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Superresolution microscopy reveals linkages between ribosomal DNA on heterologous chromosomes

The spatial organization of the nucleus is startlingly intricate. Chromosomes occupy distinct territories, and can form complex intra- and inter-chromosomal connections. However, these associations, especially those of the inter-chromosomal variety, are still mysterious in frequency and function.

A recent study out of Stowers Institute took a closer look at these inter-chromosomal networks, hoping to gain a better understanding of their purpose in the developing cell. The paper, Superresolution microscopy reveals linkages between ribosomal DNA on heterologous chromosomes, uncovered some previously unknown findings about the way interphase chromosomes interact with one another. I sat down with Dr. Tamara Potapova, one of the team’s leading researchers, to talk about what they discovered:

Me: Can you tell us a little about your research? What are your particular areas of interest?

Dr. Potapova: My personal long-term interest is in the cell cycle, chromosome biology, and genome organization, with a particular focus on understanding how alterations in chromosome number and structure arise and what consequences they have on cell and organism physiology. My lab, the Gerton Lab, has been focused on studying chromosome biology and genome integrity. Biology of ribosomal DNA (rDNA) has been an important focus of many of our studies.

Me: Can you talk about your team’s motivation behind conducting this study? As you mentioned in the intro, there hasn’t been much research on the actual effects of inter-chromosomal associations (all we know is that they exist). What made you want to take a closer look at this feature?

Dr. P: In the interphase nucleus, chromosomes are de-condensed and occupy territories that are naturally closely associated. Previously, inter-chromosomal DNA interactions have been inferred from chromosome conformation capture methods such as Hi-C. While the existence of inter-chromosomal associations was implied by these studies, the nature of the associations was unknown, as was the mode of their resolution during mitosis.

The rDNA genes presented a good model of inter-chromosomal interactions because they are present on multiple chromosomes that associate together to form the nucleolus, and because these interactions persist in mitosis we were able to actually visualize them.

(A little background on rDNA genes: these encode the rRNA components of the ribosome, and are clustered on the short arms of chromosomes 13, 14, 15, 21, and 22. Although their number varies between individuals, most people have several hundred copies. As Tara mentioned, they’re ideal for observing connections between chromosomes because they’re found at loci that often form these associations.)

Me: How did you use FISH to facilitate this study? Can you talk about the importance of the technology for being able to visualize inter-chromosomal interactions?

Dr. P: For this study, FISH was our key tool. It was very helpful to have probes that have good fluorescent labeling, particularly for structural illumination microscopy that requires high fluorescent signal intensity and high contrast. Using FISH probes that give a strong signal and low background (including the RP11-450E20 and RP23-225M6 probes provided by Empire Genomics) was very instrumental for this study.

Me: You ended up finding that these linkages were both far more frequent than previously know, and, importantly, didn’t result in any mistakes in cell division or chromosome copy number. Can you expand on the implications of these findings?

Dr. P: It was surprising how common they can be, and that their presence did not cause chromosomal mis-segregations and aneuploidy. Our findings emphasize that the genomic organization within the nucleus cannot be viewed simply as strings of genes on individual chromosomes; the spatial context is also important. Contacts between chromosomes may provide a structural basis for long-range genetic interactions, the formation of specialized compartments within the nucleus, and possibly have other functions that are currently unknown.

Me: You didn’t find these linkages to be pathological, but you do suspect they’re related to Robertsonian translocations, which can be. Do you see any value in looking into rDNA linkages as a possible precursor to pathological translocations/genetic instability?

Dr. P: Great question. Our findings suggest that transcriptionally active rDNA loci engage in physical inter-chromosomal connections that may be a normal feature of genome organization. They can, potentially, pre-dispose acrocentric chromosomes to the formation of Robertsonian fusions, but clearly most of the time they are not pathological. However, rDNA is not the only genetic locus that can form inter-chromosomal linkages, and we have preliminary data suggesting that there are other cases where inter-chromosomal linkages are highly pathological. Stay tuned for more results from our lab.

Rachel McMahon-Eagan