6 APRIL 2017 | VOL 544 | NATURE | 59
3D structures of individual mammalian
genomes studied by single-cell Hi-C
Tim J. Stevens
*, David Lando
*, Srinjan Basu
*, Liam P. Atkinson
, Yang Cao
, Steven F. Lee
, Martin Leeb
†, Kai J. Wohlfahrt
, Aoife O’Shaughnessy-Kirwan
, Julie Cramard
, Andre J. Faure
, Meryem Ralser
, Enrique Blanco
†, Miriam Sansó
, Matthieu G. S. Palayret
, Ben Lehner
, Luciano Di Croce
, Anton Wutz
†, Brian Hendrich
& Ernest D. Laue
Our understanding of nuclear architecture has been built on electron
and light microscopy studies that suggest the existence of territories
pervaded by an inter-chromosomal space through which molecules
diffuse to and from their sites of action
. In parallel, biochemical studies,
in particular chromosome conformation capture experiments (such as
3C and Hi-C) in which DNA sequences in close spatial proximity in
the nucleus are identified after restriction enzyme digestion and DNA
ligation, have provided molecular information about chromosome
. At the megabase scale, Hi-C experiments have partitioned
the genome into two (A or B) compartments
. In addition, they have
provided evidence for 0.5–1.0-Mb topological-associated domains
, as well as smaller loops (hundreds of kilobases)
experiments have further shown that enhancers make direct physical
interactions with promoters, and that these interactions are stabi-
lized by a network of protein–protein interactions involving CTCF,
cohesin and Mediator
. Although probabilistic methods can be used to
calculate ensembles of low-resolution models that are consistent with
population Hi-C data
, understanding genome structure at higher
resolution requires the development of single-cell approaches.
In mitotic cells, both TADs and A/B compartments disappear
and thus the structural complexity of interphase chromosomes is re-
established during the G1 phase. To study interphase genome structure,
we have combined imaging with an improved Hi-C protocol (Fig. 1a)
to determine whole-genome structures of single G1-phase haploid
mouse embryonic stem (ES) cells at the 100-kb scale. The structures
allow us to study TAD and loop structure genome-wide, to analyse
the principles underlying genome folding, and to understand which
factors may be important for driving chromosome/genome structure.
We also illustrate how combining single-cell genome structures with
population-based RNA sequencing (RNA-seq) and chromatin immu-
noprecipitation followed by high-throughput sequencing (ChIP–seq)
The folding of genomic DNA from the beads-on-a-string-like structure of nucleosomes into higher-order assemblies is
crucially linked to nuclear processes. Here we calculate 3D structures of entire mammalian genomes using data from a new
chromosome conformation capture procedure that allows us to first image and then process single cells. The technique
enables genome folding to be examined at a scale of less than 100 kb, and chromosome structures to be validated. The
structures of individual topological-associated domains and loops vary substantially from cell to cell. By contrast, A and
B compartments, lamina-associated domains and active enhancers and promoters are organized in a consistent way on
a genome-wide basis in every cell, suggesting that they could drive chromosome and genome folding. By studying genes
regulated by pluripotency factor and nucleosome remodelling deacetylase (NuRD), we illustrate how the determination
of single-cell genome structure provides a new approach for investigating biological processes.
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical
Campus, Cambridge CB2 0QH, UK.
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
Wellcome Trust – MRC Stem Cell Institute, University of
Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK.
EMBL-CRG Systems Biology Unit, Centre for Genomic Regulation (CRG), 08003 Barcelona, Spain.
Universitat Pompeu Fabra, 08003
Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Spain. †Present addresses: Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter,
Dr. Bohr-Gasse 9/3, 1030 Vienna, Austria (M.L.); Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Department of Human Genetics, Miami, Florida 33136,
USA (L.M.); Inst. f. Molecular Health Sciences, ETH Zurich, HPL E 12, Otto-Stern-Weg 7, 8093 Zürich, Switzerland (A.W.).
* These authors contributed equally to this work.
In-nucleus Hi-CIdentify contactsCompute structure
Cell 1 – all chromosomes
Cell 1Cell 2 Cell 3 Cell 4Cell 5Cell 6Cell 7Cell 8
Inter-chromosome contact density (% max.):
Figure 1 | Calculation of 3D genome structures from single-cell Hi-C
data. a, Schematic of the protocol used to image and process single nuclei.
b, Colour-density matrices representing the relative number of contacts
observed between different pairs of chromosomes. c, Five superimposed
structures from a single cell, from repeat calculations using 100-kb
particles and the same experimental data, with the chromosomes coloured
differently. An expanded view of chromosome 10 (Chr 10) is shown,
coloured from red to purple (centromere to telomere), together with an
illustration of the restraints determining its structure.
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