with a pitch of ~250 nm within the cylindrical
shape of chromatids (68).
Such loop arrangements can naturally emerge
as a result of a loop extrusion process. Loop
extrusion has been hypothesized as a mechanism
of chromosome compaction (69, 70) and most
recently examined by simulations (25, 48, 71) and
supported by single-molecule studies (72). In this
process, each condensin starts forming a progressively larger loop until it dissociates or stops as
it is blocked by neighboring condensins or other
DNA-binding proteins. A recent study demonstrated that this process can form an array of
consecutive loops (8) with condensins forming a
central scaffold in the middle of a cylindrical
chromosome (48), essential features of mitotic
chromosomes. Sister chromatids are resolved by
late prophase (11–13), indicating that the formation of loop arrays occurs as sister chromatid
arms become separated.
Another aspect of loop extrusion is that loop
sizes are established by a dynamic process of
condensin exchange, without a need for barrier
elements or specific loading sites (25). This is
consistent with our Hi-C data that suggests that
loop bases are not positioned at specific reproducible positions [e.g., scaffold or matrix attachment regions (73, 74)] in a population of cells.
Analysis of published ChIP data for SMC2 in
mitotic DT40 cells (45) shows a low level of condensin binding throughout the genome and only
very few loci enriched in condensin binding:
Only 289 sites show more than a fivefold enrichment compared with DNA input, and 4617 sites
show more than twofold enrichment. These numbers are much lower than the 16,000 inner loops
our data and models predict. The condensin-enriched sites show a Hi-C interaction pattern
consistent with them being at the bases of loops
slightly more frequently than other loci (fig. S13).
On the basis of these analyses, we estimate that
more than 95% of mitotic loops are not positioned
at specific loci.
Simulations show that loop extrusion slowly
approaches the steady state by exchanging
condensins and gradually increasing loop sizes
during this process (25). This is consistent with
the gradual growth of loops up to 500 kb by slowly-exchanging condensin II, as well as relatively rapid
formation of 60- to 80-kb inner loops by the
more rapidly-exchanging condensin I (75).
The formation of nested loops was critical
for our polymer simulations to reproduce pro-
metaphase Hi-C data because only this allowed
a higher linear chromatin density. In this ar-
chitecture, the outer loop bases are located at
the central scaffold, whereas the inner, nested
loop bases are radially displaced. Our analy-
sis of condensin I or II depletion reveals that
condensin II generates outer loops and con-
densin I generates inner loops. Our simulations
reveal that this nested loop arrangement can
be explained by the longer half-life of con-
densin II and the shorter half-life of condensin
I on chromatin, as measured by fluorescence
recovery after photobleaching (75) (movie S1).
Nested loops form only in prometaphase,
when condensin I gains access to chromatin.
Thus, loop extrusion models can explain the
nested loop arrangement of condensed mitotic
Why condensin II–based scaffolds acquire he-
licity only in prometaphase (and not in prophase)
is not known, but this could involve interactions
with other proteins, such as DNA topoisomerase
II alpha or KIF4A. Our estimates of the radius of
the prometaphase scaffold (30 to 100 nm) are
consistent with a 50-nm length of SMC coiled
coils that can interact with each other through
HEAT repeats (76) ,which are known for their
ability to self-assemble into a helical spiral stair-
case (77). Gradual formation of such a HEAT-
mediated staircase and binding of other factors
can explain how the pitch and the radius of the
helix increase in time.
Mitotic chromatin still condenses in the absence
of both condensin I and II, although individu-
alized rod-shaped chromosomes are not formed
and cells cannot progress into anaphase. This sug-
gests additional mechanisms by which chromatin
becomes condensed during mitosis. Our simula-
tions also show that to achieve agreement with
Hi-C data, chromatin should be condensed (com-
putationally analogous to poor solvent condi-
tions), forming densely packed chromatin loops
within mitotic chromosomes, akin to the dense
packing of chromatin observed in mitotic chro-
mosomes by electron microscopy (46, 78, 79).
The molecular basis for this compaction is not
known but may involve mitosis-specific chro-
matin modifications (80, 81) or active motor
proteins such as KIF4A (82, 83).
The chromosome morphogenesis pathway de-
scribed here, and the identification of distinct
architectural roles for condensin I and II in orga-
nizing chromosomes as nested loop arrays wind-
ing around a helical spiral staircase scaffold within
a cylindrical chromatid, can guide future experi-
ments to uncover the molecular mechanisms by
which these complexes, and other key components
such as topoisomerase II alpha and KIF4A, act in
generating, (re)arranging, and condensing chro-
matin loops to build the mitotic chromosome.
DT40 cell cultures synchronously entering mitosis
were analyzed by Hi-C, imaging, and proteomics
to determine the structure of chromosomes. Hi-C
data were used to quantify chromosome compartmentalization and to derive relationships between contact frequency P and genomic distance s.
Coarse-grained models and equilibrium polymer
simulations were performed to test models of
prophase and prometaphase chromosome organization against Hi-C data and to identify the
best-fitting parameters for the size of loops, helical
turn and pitch, and linear density (megabases per
micrometer of chromosome length). Imaging of
chromosome dimensions and condensin localization was carried out to validate model predictions.
Cell lines expressing condensin subunits fused
to auxin-inducible degron domains were used
to efficiently deplete these subunits before cells
entered mitosis. Hi-C and imaging analysis were
then performed to assess the effects of condensin
depletion on mitotic chromosome formation.
Detailed procedures for all methods are de-
scribed in the supplementary materials.
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