G2 chromosomes, which are composed of two
closely aligned and likely catenated sister chromatids, are organized similarly to G1
This interphase chromosomal organization was
rapidly lost upon release of cells into prophase. As
soon as 5 min after removal of 1NM-PP1, we detected a marked reduction in the typical plaid pattern of long-range interactions, indicating a loss of
compartments (Fig. 1B). By 10 min (late prophase),
compartments were mostly gone. At the same
time, TADs were also lost (Fig. 1C and fig. S8).
We used eigenvector decomposition to quantify the disappearance of compartments (33). The
first eigenvector readily captured compartments
at t = 0 and 2.5 min, but starting at t = 5 min it
explained progressively less of the variance in
the Hi-C interaction maps, indicating weakening of the compartment structure. By t = 7.5 min,
the strength of the first eigenvector fell to 17%
(from 80% at t = 0 min), and by t = 10 min, it no
longer captured compartments. Loss of compartments was also quantified by calculating the ratio
of A-to-A or B-to-B interactions over A-to-B interactions for the full time course. From t = 0 to
2.5 min and onward, this fraction decreased steadily, indicating that preferential interactions within
compartments are lost (Fig. 1D and fig. S9).
The strength of TADs can be quantified using
the insulation score, which indicates the amount
of contacts formed across a locus up to a certain
distance (32). TAD boundaries have a low score
(indicative of high insulation), whereas loci in-
side TADs show a high score (little insulation).
The genome-wide variance of insulation scores
provides a quantitative measure of the presence
of TADs (8). Starting at t = 2.5 min, the variance
of the insulation profiles progressively decreased,
indicating the loss of TADs (Fig. 1C and fig. S5B).
By t = 7.5 min, the variance was reduced by more
than a factor of 2, and by t = 10 min, no TADs
were detected. This conclusion was confirmed by
plotting the average Hi-C interaction pattern at
and around TAD boundaries identified in G2 at
different time points during mitosis (Fig. 1C).
Insulation was strongest in G2, and, by late pro-
phase, insulation values were near background
levels (quantified in fig. S10). We conclude that
compartments and TADs disappear rapidly dur-
ing early prophase.
By late prophase, when sister arms have resolved
(11, 34), and around the time of nuclear envelope
breakdown (t 7.5 to 10 min), the Hi-C maps are
characterized by a general decay of contact
frequency P with genomic distance s (Fig. 2A).
The shape of the P(s) curve changes as prophase
progresses. In G2 cells, it is shallow [P(s) s−0.5]
up to a distance of several hundred kilobases,
reflecting compaction within TADs (35, 36), but
for larger distances the decay becomes steeper.
During prophase, the initial shallow decay extends
for longer-range interactions, with a steeper drop
at 2 Mb at t = 10 min, which suggests a higher
degree of compaction. As we demonstrate below,
this decay and shape are consistent with the
formation of a linearly arranged, layered organization of the chromosome (8), where the size of
each layer corresponds to the position of the
steep drop in the P(s) curve.
Appearance of a second diagonal band in
Hi-C maps from prometaphase cells
At t = 15 min, when cells have entered prometaphase, the Hi-C maps produce a P(s) curve
with a drop at 2 Mb. A distinct second diagonal
band appears, running in parallel with the primary diagonal for all loci and chromosomes
(Fig. 1B and fig. S11). This second diagonal represents increased interaction frequencies between
any pair of loci separated by ~3 Mb. At 15 min,
this feature is clearly observed in P(s) plots as a
local peak at ~3 Mb (Fig. 3A and figs. S6 and S7).
As cells progress through prometaphase, the
position of the drop in P(s) and the position of
the second diagonal migrate to larger genomic
distances (Fig. 3A and figs. S6 and S7). By t =
60 min, when compact metaphase chromosomes
have formed, the second diagonal is positioned
at ~12 Mb and appears more diffuse. The second
diagonal appears in all chromosomal maps, and
Gibcus et al., Science 359, eaao6135 (2018) 9 February 2018 2 of 12
Fig. 1. Chromosome morphogenesis during synchronous mitosis.
(A) Representative DAPI images of nuclei and chromosomes in CDK1as
DT40 cells taken at the indicated time points (in minutes) after release from
1NM-PP1–induced G2 arrest show mitotic chromosome formation. NEBD,
nuclear envelope breakdown. (B) Hi-C interaction maps of chromosome 7
(binned at 100 kb) from cells collected at the indicated time points in prophase
and prometaphase show large-scale changes in contact frequencies as
cells progress through mitosis. (C) The average interaction maps centered
around G2 TAD (topologically associating domain) boundaries. TAD
boundaries disappear. (D) Compartmentalization saddle plots: average
distance-normalized interaction frequencies between cis-pairs of 100-kb
bins arranged by their G2 eigenvector value (EV1). Compartments disappear.