Chromosomes are the physical realization of genetic information and thus form the basis for its readout and propagation. Here we present a high-resolution chromosomal contact map derived from a modified genome-wide chromosome conformation capture approach applied to Drosophila embryonic nuclei. The data show that the entire genome is linearly partitioned into well-demarcated physical domains that overlap extensively with active and repressive epigenetic marks. Chromosomal contacts are hierarchically organized between domains. Global modeling of contact density and clustering of domains show that inactive domains are condensed and confined to their chromosomal territories, whereas active domains reach out of the territory to form remote intra- and interchromosomal contacts. Moreover, we systematically identify specific long-range intrachromosomal contacts between Polycomb-repressed domains. Together, these observations allow for quantitative prediction of the Drosophila chromosomal contact map, laying the foundation for detailed studies of chromosome structure and function in a genetically tractable system.

Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single cell Hi-C, combined with genome-wide statistical analysis and structural modeling of single copy X chromosomes, to show that individual chromosomes maintain domain organisation at the megabase scale, but show variable cell-to-cell chromosome territory structures at larger scales. Despite this structural stochasticity, localisation of active gene domains to boundaries of territories is a hallmark of chromosomal conformation. Single cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organisation underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns.Chromosome conformation capture 1 (3C) and derivative methods (4C, 5C and Hi-C) [2][3][4][5][6] have enabled the detection of chromosome organisation in the 3D space of the nucleus. These methods assess millions of cells and are increasingly used to calculate conformations of a range of genomic regions, from individual loci to whole genomes 3,[7][8][9][10][11] . However, fluorescence in situ hybridisation (FISH) analyses show that genotypically and phenotypically identical cells have non-random, but highly variable genome and chromosome conformations 4,12,13 probably due to the dynamic and stochastic nature of chromosomal structures [14][15][16] . Therefore, whilst 3C-based analyses can be used to estimateCorrespondence and requests for materials should be addressed to PF (peter.fraser@babraham.ac.uk) for the single cell Hi-C method, AT (amos.tanay@weizmann.ac.il) for the statistical analysis, or EDL (e.d.laue@bioc.cam.ac.uk) for the structural modelling.. Author Contributions TN and PF devised the single cell Hi-C method. TN performed single cell Hi-C and DNA FISH experiments. SS carried out ensemble Hi-C experiments. WD microscopically isolated single cells. YL, EY and AT processed and statistically analyzed the sequence data. TJS and EDL developed the approach to structural modelling and analysed X chromosome structures. TJS wrote the software for 3D modeling, analysis and visualisation of chromosome structures. TN, YL, TJS, EDL, AT and PF contributed to writing the manuscript, with inputs from all other authors.Data deposited in NCBI's Gene Expression Omnibus (Nagano et al., 2013) and are accessible through GEO Series accession number GSE48262 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE48262).The authors declare that they have no competing financial interests. an average conformation, it cannot be assumed to represent one simple and recurrent chromosomal structure. To move from probabilistic chromosome conformations averaged from millions of cells towards determinati...

Hi-C experiments measure the probability of physical proximity between pairs of chromosomal loci on a genomic scale. We report on several systematic biases that substantially affect the Hi-C experimental procedure, including the distance between restriction sites, the GC content of trimmed ligation junctions and sequence uniqueness. To address these biases, we introduce an integrated probabilistic background model and develop algorithms to estimate its parameters and renormalize Hi-C data. Analysis of corrected human lymphoblast contact maps provides genome-wide evidence for interchromosomal aggregation of active chromatin marks, including DNase-hypersensitive sites and transcriptionally active foci. We observe extensive long-range (up to 400 kb) cis interactions at active promoters and derive asymmetric contact profiles next to transcription start sites and CTCF binding sites. Clusters of interacting chromosomal domains suggest physical separation of centromere-proximal and centromere-distal regions. These results provide a computational basis for the inference of chromosomal architectures from Hi-C experiments.

To ensure proper gene regulation within constrained nuclear space, chromosomes facilitate access to transcribed regions, while compactly packaging all other information. Recent studies revealed that chromosomes are organized into megabase-scale domains that demarcate active and inactive genetic elements, suggesting that compartmentalization is important for genome function. Here, we show that very specific long-range interactions are anchored by cohesin/CTCF sites, but not cohesin-only or CTCF-only sites, to form a hierarchy of chromosomal loops. These loops demarcate topological domains and form intricate internal structures within them. Post-mitotic nuclei deficient for functional cohesin exhibit global architectural changes associated with loss of cohesin/CTCF contacts and relaxation of topological domains. Transcriptional analysis shows that this cohesin-dependent perturbation of domain organization leads to widespread gene deregulation of both cohesin-bound and non-bound genes. Our data thereby support a role for cohesin in the global organization of domain structure and suggest that domains function to stabilize the transcriptional programmes within them.Chromosomal compartmentalization has been recognized as important for genome function. High-resolution techniques such as Hi-C, ChIP- and 4C-seq offer novel insights into cohesin's dynamic role in shaping the nuclear architecture.

Recent evidence suggests that the timing of DNA replication is coordinated across megabase-scale domains in metazoan genomes, yet the importance of this aspect of genome organization is unclear. Here we show that replication timing is remarkably conserved between human and mouse, uncovering large regions that may have been governed by similar replication dynamics since these species have diverged. This conservation is both tissue-specific and independent of the genomic G+C content conservation. Moreover, we show that time of replication is globally conserved despite numerous large-scale genome rearrangements. We systematically identify rearrangement fusion points and demonstrate that replication time can be locally diverged at these loci. Conversely, rearrangements are shown to be correlated with early replication and physical chromosomal proximity. These results suggest that large chromosomal domains of coordinated replication are shuffled by evolution while conserving the large-scale nuclear architecture of the genome.