top (l to r): Yu Zhang, Ph.D., and Frederick W. Alt, Ph.D.
bottom (l to r): Yu-Jui (Ray) Ho and Frederick W. Alt, Ph.D.
How the 3D Organization of Genes Contributes to Chromosomal Rearrangements in Cancer
Researchers in the laboratory of Frederick Alt at the Program in Cellular and Molecular Medicine at Children's Hospital Boston and the Immune Disease Institute (PCMM/IDI), in collaboration with Job Dekker and colleagues in the Program in Systems Biology and Gene Function and Expression at the UMass Medical School, have determined that the spatial organization of the genome strongly influences gene translocations resulting from DNA double-strand breaks (DSBs), an important finding in the genomics of cancer.
In Cell (online February 16 and in print March 2, 2012), co-first authors Yu Zhang and Rachel Patton McCord and their colleagues report combining two recently developed high-throughput genome-wide techniques to study how the genome's physical layout contributes to chromosomal reorganizations called translocations. Applying 3D high-resolution genomic mapping to mouse cells, they found that the spatial organization of the genome contributes dramatically to the frequency with which random DSBs translocate genome-wide.
Following up several of their recent studies, the Alt lab continues to explore mechanisms responsible for generating breaks in DNA and creating genomic instability. Many of their efforts in this area have centered around the study of immune cells, in which antibody genes are mutated, broken, and joined by mechanisms that diversify the immune response. Sometimes, however, these mechanisms go awry, leading to translocations and cancer. For the last several years, the lab has shifted its focus from the translocation end products found in cancer genomes of tumor cells to the complex multi-step processes that produce them.
Cytogenetic approaches have long been used to study the individual aberrant chromosomal translocations found in tumor cells. However, the Alt lab has been interested in how translocations happen at the broader genome level.
To pursue such studies, several years ago the Alt lab developed high-throughput genome-wide translocation sequencing (HTGTS), a method for mapping all the possible rearrangements of a particular chromosome break. Created by Dr. Zhang and several Alt lab colleagues, and published in Cell last fall, HTGTS is based on generating a specific break in a given chromosome with DNA-cutting enzymes such as I-SceI and then determining the sites to which it translocates across the entire genome.
Earlier in his career, Dr. Dekker invented Chromosome Conformation Capture (3C), a technique that detects physical interactions between genomic elements. Several years ago, he and his lab developed an extension of 3C called Hi-C, which reveals how physically cross-linkable every sequence in the genome is with every other sequence. The landmark achievement of Hi-C, published in 2009, was to provide a 3D spatial organization map of the entire human genome. This map revealed that the folding patterns of a given chromosome promote a high level of interaction among many sequences along its length.
Working on several types of antibody-producing B cells in the mouse, the Alt lab noticed that translocations from a break introduced into a given chromosome seemed to go most frequently to other breaks all along that same chromosome. Based on Dekker's Hi-C findings with respect to human chromosomes, the Alt lab speculated that the apparent predominance of "intra-chromosomal" translocations might reflect 3D genome organization. Based on these observations, the Alt and Dekker labs joined forces two years ago, combining Hi-C and HTGTS to directly examine the relationship between 3D genome organization and translocations.
To derive general rules for the effects of spatial proximity on translocations resulting from both physiological and introduced breaks in various parts of the genome, Dr. Zhang's first goal was to put target break sites in many different chromosomes of B-lineage cell lines that were in the process of making chromosomal breaks to assemble antibody genes. Dr. Zhang also used cell lines that were arrested at one stage of division to avoid potential problems caused by dividing (reproducing) cells constantly undergoing genomic spatial organization change as chromosomes are replicated. Finally, to encourage chromosomal rearrangements, the team used cells deficient in the DNA damage-control protein ATM.
To examine spatial organization, Rachel McCord in the Dekker lab applied the Hi-C technology to these same cell cycle-arrested B-lineage lines to generate the highest-resolution map of the 3D organization of a mammalian genome to date. With this map and the superimposed HTGTS translocation maps generated by Dr. Zhang, the Alt/Dekker team was able to generate a 3D map of translocations from many target break sites within the genome.
Comparative analyses of the these two maps showed that regardless of their placement in the genome, introduced breaks recurrently translocated to sites of endogenous chromosome breaks formed during antibody gene assembly. This was true for 40 different pairs of such breaks, whether or not the breaks were, on average, proximal in a large fraction of cells.
These findings led to an important conceptual innovation: the consideration of cellular heterogeneity in the interpretation of the new data. Thus, although each given type of somatic cell in an animal has the same average genome organization, individual cells vary greatly in 3D genomic configuration, perhaps reflecting subtle differences in the way the genome is used in closely related cells. The principle of cellular heterogeneity predicts that even among the related skin cells of one animal (for example), it is possible that no two cells have exactly identical genome organization.
Alt's team applied the following simple reasoning regarding the frequency of breaks and joins. If two sequences break constantly but are never near each other, they won't join; if they're often near each other but never break, they won't join. These are the extremes, but the real situation is usually somewhere in between, with small differences among cells. Thus, as Zhang et al. write, cellular heterogeneity in the genome means that "translocation frequency is a function of DSB frequency at two sites and the fraction of individual cells in a population in which the sites are juxtaposed." As Dr. Alt puts it, "You don't have to think only of the sequences that are the most proximal as the ones that form recurrent translocations. If they're proximal some of the time but they break a lot, they still will frequently be driven to translocation by the high frequency of breaks."
Recurrent breaks, such as those generated during antibody gene assembly, and genomic spatial heterogeneity can lead to the types of recurrent translocations found in lymphoid tumors. However, recurrent breaks in a model system can obscure major effects of spatial organization on the translocation of other sequences where DSBs are less frequent. This is an important consideration, as many cancer-related translocations in non-lymphoid tumors involve sequences that do not necessarily break frequently.
To generate a model system that avoids the confounding effect of dominant breaks, Dr. Zhang irradiated the test cells, which produced several hundred random DSBs distributed across the genome. The result was very surprising indeed: the whole chromosome containing a target break became a hot spot for its translocation. However, because mice (like humans) have diploid DNA (two copies of each chromosome), the team could not determine whether translocations were happening on the cis chromosome (the same one where the break happened) or in trans (on the other copy). This prompted Dr. Zhang to breed a mouse in which the two homologous chromosomes are distinguishable by single-nucleotide polymorphisms (SNPs). Analyses of cells from these mice showed that the translocations occurred dominantly only in cis.
The Hi-C studies of Dr. McCord revealed that such cis translocations are promoted by the tendency of the cell cycle-arrested mouse chromosomes to be rolled into a globule, greatly increasing the probability that sequences separated by long distances along their linear length will be near each other in 3D. Further, the combination of Hi-C and HTGTS in the irradiated B-lineage cells revealed that target breaks translocated to other sequences genome-wide in a manner that was directly related to their pre-existing spatial proximity. This finding has major relevance to interpreting the mechanisms leading to translocations that occur in many different types of cancer.
Beyond the new finding regarding mechanisms that drive translocations, the current publication in Cell underscores the importance of the first 3D genomic mapping of the mouse, a widely used experimental animal, providing a rich resource for others in the field. Even given the differences between mice and humans regarding chromosomal number and centromere placement (i.e., acentric vs. dicentric), their overall genomic organization turns out to be very similar. In addition to being folded or rolled in the cellular nucleus into a globule, both genomes are divided into active and inactive compartments, and large chromosomes tend to associate more closely with other large chromosomes, and small with small.
As newcomers from more biochemical and molecular genetic approaches, the members of the Alt lab consider themselves the beneficiaries of entering a new technical genomics field at a time when a great deal of progress has recently been made by other researchers. They hope that their own contributions of new techniques, new models, and controls will prove to be useful to the field in general.
Dr. Alt sums it all up: "Now we have taken one technology, which gives you the whole 3D map of the genome, and combined it with a second one that shows you where breaks translocate within the genome. When we put them together, we learn quite a few things and gain a very helpful new perspective from which to view cancer genomes."
This study was supported by the National Cancer Institute, the National Human Genome Research Institute, the Howard Hughes Medical Institute, the Leukemia and Lymphoma Society, the W.M. Keck Foundation, the Cancer Research Institute, and the German National Merit Foundation.
Yu Zhang, Rachel Patton McCord, Yu-Jui Ho, Bryan R. Lajoie, Dominic G. Hildebrand, Aline C. Simon, Michael S. Becker, Frederick W. Alt and Job Dekker. Spatial organization of the mouse genome and its role in recurrent chromosomal translocations. Cell. 2012 Feb 16. [Epub ahead of print]