The three-dimensional organization of a genome plays a critical role in regulating gene expression, yet little is known about the machinery and mechanisms that determine higher-order chromosome structure. Here we perform genome-wide chromosome conformation capture analysis, fluorescent in situ hybridization (FISH), and RNA-seq to obtain comprehensive three-dimensional (3D) maps of the Caenorhabditis elegans genome and to dissect X chromosome dosage compensation, which balances gene expression between XX hermaphrodites and XO males. The dosage compensation complex (DCC), a condensin complex, binds to both hermaphrodite X chromosomes via sequence-specific recruitment elements on X (rex sites) to reduce chromosome-wide gene expression by half. Most DCC condensin subunits also act in other condensin complexes to control the compaction and resolution of all mitotic and meiotic chromosomes. By comparing chromosome structure in wild-type and DCC-defective embryos, we show that the DCC remodels hermaphrodite X chromosomes into a sex-specific spatial conformation distinct from autosomes. Dosage-compensated X chromosomes consist of self-interacting domains (∼1 Mb) resembling mammalian topologically associating domains (TADs). TADs on X chromosomes have stronger boundaries and more regular spacing than on autosomes. Many TAD boundaries on X chromosomes coincide with the highest-affinity rex sites and become diminished or lost in DCC-defective mutants, thereby converting the topology of X to a conformation resembling autosomes. rex sites engage in DCC-dependent long-range interactions, with the most frequent interactions occurring between rex sites at DCC-dependent TAD boundaries. These results imply that the DCC reshapes the topology of X chromosomes by forming new TAD boundaries and reinforcing weak boundaries through interactions between its highest-affinity binding sites. As this model predicts, deletion of an endogenous rex site at a DCC-dependent TAD boundary using CRISPR/Cas9 greatly diminished the boundary. Thus, the DCC imposes a distinct higher-order structure onto X chromosomes while regulating gene expression chromosome-wide.
Crane, E., Bian, Q., McCord, R.P., Lajoie, B.R., Wheeler, B.S., Ralston, E.J., Uzawa, S., Dekker, J., and Meyer, B.J. (2015). Condensin-driven remodelling of X chromosome topology during dosage compensation. Nature 523, 240-244.
Chromosome conformation capture (3C) has revolutionized the ways in which the conformation of chromatin and its relationship to other molecular functions can be studied. 3C-based techniques are used to determine the spatial arrangement of chromosomes in organisms ranging from bacteria to humans. In particular, they can be applied to the study of chromosome folding and organization in model organisms with small genomes and for which powerful genetic tools exist, such as budding yeast. Studies in yeast allow the mechanisms that establish or maintain chromatin structure to be analyzed at very high resolution with relatively low cost, and further our understanding of these fundamental processes in higher eukaryotes as well. Here we provide an overview of chromatin structure and introduce methods for performing 3C, with a focus on studies in budding yeast. Variations of the basic 3C approach (e.g., 3C-PCR, 5C, and Hi-C) can be used according to the scope and goals of a given experiment.
Belton, J.M., and Dekker, J. (2015a). Chromosome Conformation Capture (3C) in Budding Yeast. Cold Spring Harbor protocols 2015, pdb prot085175.
Belton, J.M., and Dekker, J. (2015b). Chromosome Conformation Capture Carbon Copy (5C) in Budding Yeast. Cold Spring Harbor protocols 2015, pdb prot085191.
Belton, J.M., and Dekker, J. (2015c). Hi-C in Budding Yeast. Cold Spring Harbor protocols 2015, pdb prot085209.
Belton, J.M., and Dekker, J. (2015d). Measuring Chromatin Structure in Budding Yeast. Cold Spring Harbor protocols 2015, pdb top077552.
Belton, J.M., and Dekker, J. (2015e). Randomized ligation control for chromosome conformation capture. Cold Spring Harbor protocols 2015, pdb prot085183.
Mitotic chromosomes are among the most recognizable structures in the cell, yet for over a century their internal organization remains largely unsolved. We applied chromosome conformation capture methods, 5C and Hi-C, across the cell cycle and revealed two distinct three-dimensional folding states of the human genome. We show that the highly compartmentalized and cell type-specific organization described previously for nonsynchronous cells is restricted to interphase. In metaphase, we identified a homogenous folding state that is locus-independent, common to all chromosomes, and consistent among cell types, suggesting a general principle of metaphase chromosome organization. Using polymer simulations, we found that metaphase Hi-C data are inconsistent with classic hierarchical models and are instead best described by a linearly organized longitudinally compressed array of consecutive chromatin loops.
Naumova, N., Imakaev, M., Fudenberg, G., Zhan, Y., Lajoie, B.R., Mirny, L.A., and Dekker, J. (2013). Organization of the mitotic chromosome. Science 342, 948-953.