The study of higher order genomic structure using novel chromosome conformation capture techniques is an important growth area of biological research. These methods are being used to study long-range interactions between or within chromosomes, and promise to elucidate the spatial organisation of the genome, and it’s functional significance. One such technique, Hi-C, which allows the identification of chromatin interactions across the entire genome, is used in a recent paper to discover that mammalian chromosomes are divided into highly self-interacting ‘topological domains’.
Hi-C works by purifying chromosomal interactions and then sequencing the products. Briefly, this is achieved by chromosomes first being cross-linked by treatment with formaldehyde; the DNA is then chopped up and the ends of the fragments are chemically marked; the fragments are then ligated together under conditions that favour ligation of cross-linked fragments. Thus the ligation products were originally in close proximity to each other. After shearing, the marked fragments are purified, and the resulting library of interacting fragments is ‘massively parallel sequenced’. Upon alignment with a reference genome sequence, one can construct a genome-wide contact matrix.
Dixon et al. applied Hi-C to mouse ES cells, human ES cells, human fibroblasts, as well as using data from mouse cortex. They found that when they analysed their data at a resolution of less than 100kb, highly self-interacting regions emerged. For example, in mouse ES cells, 2,200 of these ‘topological domains’, with a median size of 880kb, occupied ~91% of the genome. The topological domains were separated by short segments in which chromatin interactions ended abruptly, termed ‘topological boundary regions’. Interestingly, in general, the boundary regions remained the same between embryonic stem cells and differentiated cells, in both mouse and human. Hence, the overall domain architecture is generally unchanged between cell types. Surprisingly, there was also quite a high degree of conservation of boundary zones between human and mouse.
These boundary zones seem to correspond to insulator or barrier elements that are known to divide different chromatin domains, and prevent heterochromatin from spreading. For instance the HoxA locus is divided into two compartments by a known insulator element, which was found to be a topological boundary region in both human and mouse. Dixon et al. also found that the distribution of the heterochromatin associated histone modification H3K9me3 was segregated at boundary regions in differentiated cells. As the topological domains generally remain constant between stem and differentiated cell types, the boundaries seem to pre-mark the end points for heterochromatic spreading during cellular differentiation. Likewise, this shows that the topological domains are not a consequence of heterochromation formation.
In agreement with the linkage of boundary zones to insulator elements, Dixon et al found that they were enriched for binding-sites for the insulator protein CTCF. However, only 15% of global CTCF binding sites were in boundary zones, suggesting a more complex composition and function for the boundary zones. Looking at the distributions of other cellular factors, the researchers showed that boundary zones are associated with high levels of transcription; being enriched for transcription start sites, housekeeping genes, and promoter associated histone marks. Interestingly they also observed an enrichment for SINE retrotransposons. This is in agreement with a recent paper (that I wrote about) linking SINEs to the genomic spread of CTCF binding sites during evolution.
The discovery that the genome is partitioned into these topological domains is part of a growing literature dissecting genomic macro-structure. Dixon et al. compared topological domains with various other recently defined higher order levels of genomic organisation; ‘A+B’ compartments (Lieberman-Aiden et al.), lamina-associated domains, replication time zones, and large organised chromatin K9 modification domains. They concluded that topological domains are related to, but independent from each of these previously characterised architectures. This list gives one some idea of the complexity, and our shallow understanding of, higher order genomic structure. However, this tranche of new chromosome capture techniques, combined with methods for high throughput analysis of chromatin composition, are yielding a wealth of data. In the next few years we should have a far more nuanced and complete appreciation of the interplay between chromosomal architecture, chromatin state and genetic regulation. A mouth-watering prospect.
Dixon, J., Selvaraj, S., Yue, F., Kim, A., Li, Y., Shen, Y., Hu, M., Liu, J., & Ren, B. (2012). Topological domains in mammalian genomes identified by analysis of chromatin interactions Nature, 485 (7398), 376-380 DOI: 10.1038/nature11082
Lieberman-Aiden, E., van Berkum, N., Williams, L., Imakaev, M., Ragoczy, T., Telling, A., Amit, I., Lajoie, B., Sabo, P., Dorschner, M., Sandstrom, R., Bernstein, B., Bender, M., Groudine, M., Gnirke, A., Stamatoyannopoulos, J., Mirny, L., Lander, E., & Dekker, J. (2009). Comprehensive Mapping of Long-Range Interactions Reveals Folding Principles of the Human Genome Science, 326 (5950), 289-293 DOI: 10.1126/science.1181369