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A novel chromatin tether domain controls 
topoisomerase lla dynamics and mitotic 
chromosome formation 

Andrew B. Lane, Juan F. Gimenez-Abian, and Duncan J. Clarke 

^rtment of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455 

DNA topoisomerase lla (Topo lla) is the target of 
an important class of anticancer drugs, but tumor 
cells can become resistant by reducing the asso- 
ciation of the enzyme with chromosomes. Here we describe 
a critical mechanism of chromatin recruitment and exchange 
that relies on a novel chromatin tether (ChT) domain and 
mediates interaction with histone H3 and DNA. We show 

that the ChT domain controls the residence time of Topo 
lla on chromatin in mitosis and is necessary for the for- 
mation of mitotic chromosomes. Our data suggest that the 
dynamics of Topo lla on chromosomes are important for 
successful mitosis and implicate histone tail posttransla- 
tional modifications in regulating Topo lla. 


In preparation for chromosome segregation, fibers of interphase 
chromatin are remodeled to form rod- shaped chromonemas 
of mitotic chromosomes (Swedlow and Hirano, 2003; Eltsov 
et at., 2008; Nishino et al, 2012). This dramatic transformation of 
interphase chromatin to a set of physically tractable condensed 
chromosomes is complete within minutes, yet must achieve not 
just linear compaction, but also individualization of each chro- 
mosome (Gimenez-Abian et al., 1995) and resolution of the two 
sisters within the pair (Sumner, 1991). The extreme fidelity with 
which cells carry out this process of mitotic chromosome forma- 
tion is essential for preventing chromosome segregation errors. 

Chromosome morphological changes in mitosis have been 
suggested to depend on the reorganization of chromatin on a 
proteinaceous axial core, first revealed in electron micrographs 
of dehistonized condensed chromosomes (Paulson and Laemmli, 
1977; Mullinger and Johnson, 1979). Although the "axial core" 
is a cytologically defined structure, it likely corresponds to the 
chromosome scaffold, a highly stable structure that remains in- 
tact after treatment of chromosomes with micrococcal nuclease 
and 2 M NaCl. This biochemical fraction contains DNA topoi- 
somerase lla (Topo lla) and 13S condensin (Adolph et al., 1977; 
Earnshaw et al., 1985; Gasser and Laemmli, 1987), enzymes 
that function in mitotic chromosome formation. In mitosis, 
Topo lla is largely restricted to the axial core (Tavormina et al., 
2002; Maeshima and Laemmli, 2003), and the residence time of 

Correspondence to Duncan J. Clarke: clarkl 

Abbreviations used in this paper: ChT, chromatin tether; CTR, C-terminal region; 
Topo II, topoisomerase II; WT, wild type. 

Topo lla on chromosomes is very short (~15 s) in live cells 
(Tavormina et al., 2002). However, little is known about the 
mechanism that localizes Topo lla to chromosomes, and it is 
not known if the highly dynamic property of the enzyme is bio- 
logically important. 

Previous studies raised the possibility that there are dis- 
tinct factors conferring Topo lla localization upon the axial core. 
In either Drosophila melanogaster or chicken cells depleted of 
condensin, Topo lla is targeted to mitotic chromosomes but 
core enrichment is abolished (Coelho et al, 2003; Hudson et al., 
2003). This function of condensin involves its ability to gener- 
ate positively supercoiled DNA, the preferred topological sub- 
strate of Topo lla (Kimura and Hirano, 1997; McClendon et al., 
2008). It is not known if the chromosome core region is enriched 
with DNA in a positively supercoiled topological state, but this 
can be inferred from the fact that condensin localization is 
mostly restricted to the core region of chromosomes (Maeshima 
and Laemmli, 2003; Ono et al., 2003). These data are therefore 
consistent with a multi-mechanism process in which, indepen- 
dent of condensin, Topo lla can bind to chromatin but, influ- 
enced by condensin activity, Topo lla becomes enriched at the 
axial core. 

The enzyme activity of Topo lla is to perform a strand 
passage reaction that allows transit of one double helix of DNA 

© 201 3 Lane et al. This article is distributed under the terms of an Attribution-Noncommercial- 
Share Alike-No Mirror Sites license for the first six months after the publication date (see After six months it is available under a Creative Commons 
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The Rockefeller University Press $30.00 
J. Cell Biol. Vol. 203 No. 3 471-486 1 0. 1 083/jcb.201 303045 

JCB 471 

Figure 1 . Topo Ma localizes to the axial core 
of M. muntjak mitotic chromosomes. (A) GFP- 
human Topo Hot transfected into /VI. muntjak cells 
does not coimmunoprecipitate with endogenous 
M. muntjaklopo lla when precipitated using an 
anti-GFP antibody (right). GFP-human Topo Ha 
ACTR (i.e., amino acids 1-1,321] transfected 
into HeLa cells and immunoprecipitated in the 
same way coprecipitates the endogenous, 
untagged protein (left). (The anti-Topo Ha anti- 
body used for Western blotting recognizes only 
WT human or M. muntjak Topo lla and not 
Topo lla ACTR.) (B) Human Topo lla localization 
in M. muntjak cells transfected with a construct 
encoding GFP-Topo lla, fixed and stained with 
DAPI, demonstrating densely punctate localiza- 
tion of GFP-Topo lla to the axial core of mitotic 
chromosomes (bottom) and nuclear localization 
during interphase (top). Bars, 10 urn. (See also 
Video 1 .) (C, left) Enlarged portion of region 
marked by the broken-line box in B to show 
apparent coiling of the axial core. (C, right) A 
representative (more than three experimental 
repeats) plot across mitotic chromosome arms 
showing that the GFP signal (Topo lla) occupies 
a narrower region than the DNA (DAPI) signal 
(corresponds to the solid boxed region in B). 
Bars, 1 pm. 


*• MW 

HeLa (Human) (kd) 


No transfection 


Input IP:a-GFP 

hTopo lla 

(Indian Muntjac) (kD) 
No transfection 


Input IP: a-GFP 





M. muntjak Topo lla 

^M. muntjaklopo lla 





DAPI — GFP-Topolla 

< 2000 

§ 1000 
1 500 

0 12 3 
Profile position (pm) 

through another, allowing the removal of entanglements, super- 
coils, and catenations. It achieves this by making a transient 
double-strand break in one helix, passing a second helix through 
the break, then re-ligating the first (Wang, 2002). Strikingly, 
however, the domain of the enzyme sufficient for this reaction 
in vitro is not sufficient for localizing Topo Hot to chromosomes 
in cells (Linka et al., 2007). In fact, human cells contain two 
genetically distinct isoforms of Topo II (a and p) that have indis- 
tinguishable catalytic cycles, but only Topo Hot is localized to 
mitotic chromosomes, a property conferred by its divergent 
C-terminal region (CTR; Linka et al., 2007). As suggested by 
their respective localization patterns, Topo Hot is essential for 
chromosome condensation and segregation, whereas Topo lip is 
dispensable (Grue et al., 1998; Sakaguchi and Kikuchi, 2004). 

Here we describe a novel element in the CTR that dictates 
the dynamics of Topo Hot on chromosomes and is required for 
mitotic chromosome formation. We refer to this component as 
the chromatin tether (ChT) domain because it facilitates stable 
binding to chromatin. In vitro, the ChT domain is required for 
both interaction with DNA and histone H3 N-terminal tails, the 
latter being enhanced by methylation of Arg 26 and Lys 27 and 
inhibited by phosphorylation of Ser 28. To probe the biological 
importance of the ChT domain, we established a knockdown/ 

rescue system for functional analysis of Topo Ilct mutants in 
human cells and used this system to determine whether the ChT 
domain is essential for normal mitotic chromosome formation. 


Human Topo lla is enriched within the axial 
core of Muntiacus muntjak chromosomes 

To determine the mechanism of Topo lla localization to mitotic 
chromosomes in intact mammalian cells, we used a previously 
characterized human fusion protein (GFP-hTopo lla; Tavormina 
et al., 2002) and expressed it in M. muntjak cells. These cells 
are well suited to this study because M. muntjak has the largest 
mammalian chromosomes and the lowest diploid chromosomal 
number in mammals (2n = 6, females), facilitating detailed visu- 
alization of individual arms and centromeres. Further, we found 
that human Topo lla cannot be immunoprecipitated with the 
M. muntjak orthologue, which indicates that these interspecies 
isoforms cannot dimerize (Fig. 1 A). This was important because 
type II topoisomerases function as stable homodimeric enzymes 
(Tennyson and Lindsley, 1997), raising the concern that a trans- 
fected mutant Topo lla would dimerize with the endogenous 
wild-type (WT) protein and localize passively, masking effects 

JCB • VOLUME 203 • NUMBER 3 • 2D1 3 

A C 

89 90 92 



i ^^^^^^^^^^^^^^^^^^^^^^^^^m GFP-Topo lia 

^=^^^^^^^^^^^Hn GFP-Topo lla-K3R 

i w^^^^m mCherry-Topo Ha CTR 
^^^^^^H mCherry-Topo lla CTRA31 

of mutations. We thus analyzed mutant transfected forms of 
GFP-hTopo Ilct that apparently localized independently of the 
WT endogenous M. muntjak protein. 

Transfected GFP-hTopo Ilct was primarily nuclear in inter- 
phase M. muntjak cells (Fig. 1 B, top). In mitotic cells, GFP- 
hTopo lla decorated the length of chromosomes with a distinct 
enrichment at centromeres and at the axial core (Fig. 1 B, bottom; 
and Fig. 1 C). In some regions, apparent coiling of the core was 
observed (Fig. 1 C and Video 1). The enhanced localization at the 
core was evident as a structure narrower than the full width of the 
mitotic chromosome (Fig. 1 C, right). This matches the pattern 
seen in other mammalian cells when observing GFP-Topo lla 
directly or using anti-Topo Ilct antibodies against the endoge- 
nous protein (Earnshaw et al., 1985; Gimenez-Abian et al., 1995; 
Tavormina et al., 2002). The heterologous system using M. munt- 
jak cells is thus ideal for determining the mechanism of Topo Ilct 
recruitment to mitotic chromosomes. 

Topo lla associates with mitotic 
chromosomes independently of nuclear 
localization during S phase 

Based on results in the Xenopus laevis egg extract system (Cuvier 
and Hirano, 2003), the mechanism of Topo Ilct recruitment to 
mitotic chromosomes appears to depend on a prior association 
with chromatin in S phase, during DNA replication. For example. 

the protein may acquire a specific posttranslational modifica- 
tion while on interphase chromatin that licenses the later bind- 
ing to mitotic chromosomes. Indeed, previous studies have shown 
that mouse Topo Ilct must be sumoylated for mitotic kineto- 
chore localization (Dawlaty et al., 2008). 

In intact mammalian cells, the nuclear envelope breaks 
down at M phase. We reasoned that an NLS mutant of Topo Ilct 
would be cytoplasmic in S phase, but would have the opportunity 
to associate with chromosomes during M phase. Furthermore, 
the presence of endogenous M. muntjak Topo Hot in interphase 
would allow normal assembly of the chromosome axial core 
in mitosis. 

Near the C terminus of Topo Ilct, a cluster of basic resi- 
dues (aa 1,490-1,492) has been reported to be part of a func- 
tional bipartite NLS (Mirski et al., 1997; Wessel et al., 1997; 
Fig. 2 A, red text). Because NLS sequences can rely on stretches 
of positively charged residues (Conti et al., 1998; Cokol et al., 
2000; Nitiss, 2009), we generated a triple mutant at lysines 1,489, 
1,490, and 1,492, substituting with the conservative change to 
arginine that presumably minimizes changes to the overall struc- 
ture of the protein. This mutant (Topo Hct K3R) was indeed defec- 
tive for nuclear import, remaining predominantly cytosolic in 
interphase (Fig. 2 B). 

Despite the lack of access to replicating chromatin in 
S phase, GFP-hTopo Ilct K3R relocalized from the cytosol 

l GFP-Topo lla 
GFP-Topo lla-A31 







L ..J 







a> 40 
~~ 20 


0 10 


30 40 

Profile position (pixels) 

0 10 20 30 
Profile position (pixels) 

Figure 2. Nuclear localization of Topo lla during interphase is not necessary for localization to chromosomes in mitosis. (A, top) Sequence of the extreme 
CTR of Topo lla. Red residues indicate putative constituents of the bipartite NLS. Blue residues indicate the ChT domain described herein. (A, bottom) 
Schematic showing GFP- and mCherry-tagged WT Topo lla and mutants and truncation constructs used herein. GFP-Topo lla K3R comprised of K1489, 
K1490, and K1492 mutated to Arg. (B) /VI. muntjak cells transfected with GFP-Topo lla or GFP-Topo lla K3R imaged in live interphase cells. The WT 
protein is predominantly nuclear; the K3R mutant is predominantly cytoplasmic. Bars, 1 0 urn. (C) GFP-Topo lla K3R localizes similarly to WT GFP-Topo lla 
in live mitotic M. muntjak cells. Lines indicate locations of line profile plots in Fig 2 D. See also Video 2. Bars, 1 0 urn. (D) Quantification of WT Topo lla 
and Topo lla K3R abundance at mitotic chromosome cores in live M. muntjak cells. Both localize to the chromosome core with equal intensity. Shown are 
the mean of maximum intensities across n = 73 (K3R) and n = 53 (WT) core regions after background subtraction. P-value indicates two-tailed unpaired 
samples f test. Error bars indicate SD. 

Mechanism of topoisomerase II chromatin localization and dynamics • Lane et al. 473 

to chromosomes upon nuclear envelope breakdown (Video 2). 
We quantified chromosomal localization in live cells and found 
that Topo 11a K3R localized to axial cores during mitosis with 
abundance similar to that of the WT protein (Fig. 2, C and D). 
This argues against the hypothesis that the presence of Topo Hot 
in the nucleus during interphase is critical for its chromosomal 
localization in mitosis (Wang, 2002; Cuvier and Hirano, 2003). 
Similarly ruled out are mechanisms wherein passage through 
the nuclear pore during interphase is a necessary step for chro- 
mosome localization in mitosis, e.g., via posttranslational mod- 
ification by the nuclear pore SUMO ligase RanBP2 (Dawlaty 
et al., 2008). 

The CTR of Topo Ila mediates 
chromatin association 

Because the data did not support a mechanistic link to DNA 
replication or transit through nuclear pores, we sought to iden- 
tify residues of Topo Ila that are required for mitotic chromo- 
some localization. 

Linka et al. (2007) have shown that the C-terminal 211 
residues of hTopo Ila (1,321-1,531) are sufficient to direct lo- 
calization of a fluorescent protein to mitotic human chromo- 
somes. However, it is not known if this fragment can bind to the 
endogenous full-length human Topo Ila and be carried to chro- 
mosomes passively. We fused this region, referred to as the CTR 
of hTopo Ila, to mCherry and expressed it in M. muntjak cells. 
We found that mCherry-CTR localized efficiently to mitotic 
M. muntjak chromosomes (Fig. 3 A). Although the CTR was en- 
riched at the centromere regions, like full-length Topo Ila, the 
CTR occupied a broader localization on the chromosome arms, 
rather than being enriched at the axial core. This indicates that 
the CTR mediates a more broad interaction with chromosomes, 
similar to the observed localization of full-length Topo Ila seen in 
the absence of condensin (Coelho et al., 2003). Together, the data 
suggest that the CTR could provide a condensin-independent 
mechanism of chromatin association. Strikingly, a truncated 
CTR protein lacking only the extreme C-terminal 31 residues 
of the Topo Ila CTR (CTRA31) showed only minimal enrich- 
ment on mitotic chromosomes, which indicates that these are 
key residues that facilitate chromatin association (Fig. 3 A). 

Topo Ila CTR binds to DNA in vitro with 
an affinity similar to HI isoforms 

We next sought to determine the physical mechanism of associ- 
ation between the Topo Ila CTR and chromatin, and first asked 
if recombinant Topo Ila CTR fragments bind directly to DNA: 
either plasmid DNA or 60-bp oligonucleotides (Fig. 3, B-D). 

To investigate binding of supercoiled and relaxed plasmid 
DNA, we used the electromobility shift assay (EMSA) in aga- 
rose gels (Fig. 3 B). Purified recombinant Topo Ila CTR frag- 
ments or purified histone Hl° was incubated with pUC19 DNA 
containing a mixture of supercoiled, open circular, and linear 
forms. An electromobility shift was seen in all three forms of 
DNA, decreasing in magnitude as the molar ratio of Topo Ila CTR 
protein to DNA decreased (Fig. 3 B, right). Topo Ila CTRA31 
lacking the last 3 1 residues of the CTR showed a decrease in the 
magnitude of the mobility shift, which was particularly evident 

between 12.5 and 25 pmol of protein. Importantly, at 25 pmol 
of protein, the DNA shifted by Topo Ila CTR largely remained 
as a tight band, which indicates a maintained interaction with 
DNA throughout the electrophoretic migration. In contrast, smear- 
ing through the lane was observed in the case of the truncated 
mutant. This demonstrates that the CTRA3 1 protein dissociated 
from the plasmid during electrophoresis (Tseng et al., 1999; 
Kaer et al., 2008; Park et al., 2008). Compared with histone HI 0 , 
the Topo Ila CTR required ^6-12-fold more protein to pro- 
duce a shift. However, HI subtypes vary widely in their affinity 
for nonchromatinized DNA, with Hl° having up to 19-fold higher 
affinity than Hla (Orrego et al., 2007). Thus, the DNA affinity of 
the Topo Ila CTR is in a similar range to histone HI isoforms. 

To gain a relative quantification of the interaction between 
DNA and the Topo Ila CTR versus truncated versions, we used 
a pull-down strategy in which recombinant Topo Ila CTR frag- 
ments were incubated with 60-bp oligonucleotide-coated beads. 
The Topo Ila CTR, bound to the DNA beads, was consistently 
recovered using this assay (Fig. 3, C and D). However, Topo Ila 
CTR mutants truncated by 31 or 52 residues (CTRA31 and 
CTRA52) both showed a reduction in DNA binding efficiency 
(Fig. 3, C and D). Because these two mutants were precipitated 
with the DNA beads with similar efficiencies, this suggests that 
the K3 lysine residues within the bipartite NLS are not impor- 
tant for the interaction with DNA, as those residues are lacking 
in the CTRA52 mutant. Furthermore, a recombinant fragment 
of the CTR of Topo lip (Topo up 1359 1621 ) w j t h the highest homol- 
ogy to the Topo Ila CTR and which does not show strong chromo- 
somal localization in live cells (Linka et al., 2007) was recovered 
with only 10% of the efficiency of the Topo Ila CTR. This indi- 
cates that DNA binding may be a property of the a-CTR, not shared 
by the p-CTR, which promotes association with chromatin. 

Together with the in vivo experiments in M. muntjak cells, 
these data suggest that the extreme 31 residues of the CTR con- 
tain residues that confer chromatin association via stable bind- 
ing to DNA. 

Topo Ila CTR binds histone H3 in vitro 

We next considered whether the Topo Ila CTR interacts with 
chromatin proteins in addition to binding DNA. To address this, 
we used anti-HIS magnetic beads to which HIS-tagged Topo Ila 
CTR fragments were bound. We incubated these "bait" beads 
with stringently nuclease-treated HeLa cell lysates in a pull-down 
assay. Silver-staining of the resulting SDS-PAGE gels revealed 
a low-molecular weight protein at ^45 kD that was only visible 
when the Topo Ila CTR was used as bait (Fig. 4 A), but not in a 
bead-only control (mock) or in samples using a truncated CTR 
fragment (Topo Ila CTRA71). 

Given the apparent molecular weight of this coprecipitated 
protein, we performed Western blots on the pull-down samples 
and immunoblotted with antibodies recognizing histone pro- 
teins, revealing that histone H3 is specifically present in samples 
in which Topo Ila CTR was used as bait, and not in samples 
incubated with beads only (Fig. 4 B). Strikingly, H3 was not 
recovered in samples using truncated fragments of the CTR in 
which as few as 1 1 residues had been removed from the C termi- 
nus (CTRA31 and CTRA11). The inclusion of nucleases in the 

474 JCB • VOLUME 203 • NUMBER 3 • 2D1 3 






S 1.20 

<f> 0.80 


Histone H1° 

Recombinant Topo lla fragment 



M Kb 

DNA substrate: 
0.08 pmoles pUC19 

relaxed circles i 
linear i 

supercoiled i 

pmoles protein 0 
Molar ratio ° 

2 1 .5 

4? tf> J? 

0 100 50 25 12.5 0 100 50 25 12.5 0 

O JPb & nO jSJ O ^ A A rfi o 

.<*} «* /v- <P n> 

Figure 3. The C-terminal 31 residues of Topo 
lla are important for CTR chromosomal asso- 
ciation and association with DNA in vitro. 

(A) Ail. muntjak cells transfected with mCherry- 
Topo lla CTR (Topo lla residues 1,321-1,531] 
or mCherry-Topo lla CTRA3 1 (Topo lla residues 
1,321-1,500) imaged in live metaphase cells. 
mCherry-Topo lla CTR localizes to mitotic chro- 
mosomes, whereas mCherry-Topo lla CTRA31 
is localized diffusely through the nucleoplasm. 
(A, right] A representative quantification (>3 
experimental repeats) of mCherry signal across 
mitotic chromosomes (the broken lines in the 
images). Bars, 5 urn. (B) The CTR of Topo lla 
binds to plasmid DNA in vitro. EMSA analy- 
sis of supercoiled, linear, and relaxed pUC19 
DNA mixed with recombinant histone Hl° or 
CTR fragments of Topo lla (defined in A) and 
resolved on agarose gels. M = 1 kb ladder. 
The star indicates the lane where the H 1 °— 
DNA complex is assumed to be net positively 
charged and has reversed its migration direc- 
tion. Brackets highlight the mobility shifts in the 
12.5-25 pmol range for CTR and CTRA31 . 
(C) Schematic showing recombinant Topo lla 
fragments used in DNA-beads binding as- 
says (D). (D, left) Anti-His tag immunoblot 
of Topo lla fragment pull-downs using DNA- 
coated beads and purified HIS-tagged Topo lla 
CTR fragments. (D, right) Quantification of pull- 
down efficiency when uncoated bead back- 
ground is subtracted (percentage of input). 
Topo lla CTRA31 and Topo lla CTRA52 bind 
DNA with reduced efficiency. The CTR of Topo lip 
(pCTR, residues 1,359-1,621) has negligible 
DNA-binding activity, n = 3. Error bars indicate 
SD. n.s., not a statistically significant difference. 





□ 6xHis, T7 tag 
I K3 


Beads Beads 

only +DNA 


H s 





p < 0.02 

only +DNA only +DNA 

* , ^ 4 MW 

Input ^ Input ^ (k[)a) Input 

aCTR aCTRA31 aCTRA52 3CTR (1359-1621) aCTR aCTR aCTR BCTR 

A31 A52 (1359- 


lysis buffer indicates that the interaction with H3 is unlikely to 
be mediated by DNA. More importantly, recombinant CTRA31 
did interact with DNA in vitro (with ^60% of the efficiency of 
the full-length CTR; Fig. 3 D) but was not able to precipitate 
any detectable H3 from lysates. Thus, interactions with DNA 
and with H3 are likely to be independent of each other. To test 
this directly, we mixed recombinant histone H3 subtypes with 
the recombinant HIS-tagged CTR, revealing that both H3.1 and 
H3.3 coprecipitate with the CTR protein (Fig. 4 C). 

There is a wealth of evidence to suggest that histone- 
interacting proteins primarily recognize histone N-terminal tails 
(Ruthenburg et al., 2007). Further, because H3 tails are extensively 

posttranslationally modified, we were interested in the possibility 
that the Topo Hot CTR may bind in an H3 tail modification- 
specific manner. To test this, we incubated a histone H3 tail 
peptide array with purified recombinant Topo lla CTR. The 
array contained H3 tail peptides with combinations of posttrans- 
lational modifications, spotted onto a glass surface. After incu- 
bation with Topo lla CTR and extensive washing, we detected 
bound 6xHis-tagged CTR with anti-HIS antibody. Four replicates 
of the array gave similar results, and no signal was observed 
when the array was incubated with antibody alone. This analy- 
sis revealed a profile of interaction between the CTR and H3 tail 
isoforms (Fig. 4 D). Binding was enhanced by specific methylation 

Mechanism of topoisomerase II chromatin localization and dynamics • Lane et al. 475 




Anti-HIS IP from HeLa cell extract: 
silver-stained gel 

Bait only 



< CTRA71 





Anti-HIS IP from HeLa cell 
extract: Western blot 







25 > 

Co-precipitation of 
recombinant H3 subtypes 

100ng 100ng 
H3.1 H3.3 



25 > 

aHIS pulldown 




Figure 4. The Topo lla CTR binds to the N-terminal tail of histone H3 in vitro. (A) Silver-stained SDS-PAGE gel reveals an ~15-kD protein (asterisk) copre- 
cipitated from HeLa lysates with HIS-tagged Topo Ha CTR (bait). The low-molecular weight protein was not precipitated in samples using HIS-Topo Ha 
CTRA71 . (B) Immunoblot detecting immunoprecipitated recombinant HIS-Topo lla CTR fragments (top) and coprecipitated histone H3 from HeLa cell 
lysates (bottom). Topo lla CTR, but not Topo lla Al 1 or A3 1 , copurifies histone H3. Note that the data for the left three control lanes are presented again in 
Fig. S4 A. (C) Silver-stained protein gel detecting recombinant histone H3 subtypes (H3.1 and H3.3) coprecipitated with recombinant HIS-Topo lla CTR 
using anti-HIS beads. (D) Topo lla CTR binding to a peptide array containing histone H3 residues 16-35 with combinatorial posttranslational modifica- 
tions (as indicated at the bottom; R, Arg; K, Lys; S, Ser; mel, mono-methylated; me2, di-methylated; me3, tri-methylated; p, phosphorylated). Purified 
recombinant HIS-Topo lla CTR was detected strongly at spots (beneath the histograms) containing specific isoforms methylated at R26 and K27, but was 
not detected when S28 was also phosphorylated. Histograms show signals measured as integrated intensity of spots, subtracting local background. Error 
bars indicate ±SEM; n = 4. Asterisks indicate a significant difference (P < 0.01) compared with the mean of single-modified peptides. Fig. SI shows an 
expanded array. (E) Detection of recombinant Topo lla CTR coprecipitated with biotinylated histone H3 N-terminal tail (aa 21-44) peptides, either unmodified 
(Unmod), mono-methylated at Lys 27 (K27mel), tri-methylated at Lys 27 (K27me3), or phosphorylated at Ser 28 (S28p). CTR and peptides were mixed 
then precipitated with Streptavidin beads, n = 3. Error bars indicate SD. (F) ELISA assay showing binding of HIS-Topo lla CTR to immobilized H3 (21-44) 
peptides, fit to a 4 parameter logistic (4PL) curve. Note that H3 (21-44) S28p binding fits the 4PL curve poorly, which suggests a low specificity of bind- 
ing. A representative example from three experimental repeats is shown. The x axis is a log scale, except to the left of hash marks to allow inclusion of 
0 nM HIS-Topo lla data points on the same plot. 

modifications at Arg 26 and Lys 27, and was inhibited by phos- 
phorylation at Ser 28. 

To confirm the peptide array data using a more quan- 
tifiable approach, we used biotin-labeled H3 tail peptides at 
concentrations determined accurately by mass spectrometry. 
After mixing the tail peptides with recombinant CTR protein, 
we precipitated the peptides with Streptavidin beads and esti- 
mated the amount of coprecipitated CTR on protein gels. We 
observed a similar trend to that seen in the peptide arrays, where 
methylated Lys 27 isoforms preferentially coprecipitated the 
CTR, whereas Ser 28 phosphorylation had an inhibitory effect 

(Fig. 4 E). ELISA analysis of binding to the same peptides indi- 
cated that HIS-Topo lla CTR binds robustly to unmodified and 
both K27mel and K27me3 isoforms of H3 (peptides 21^14). 
In particular, the affinity of K27mel -modified peptides was 
slightly higher than that of unmodified peptide. Strikingly, HIS- 
Topo lla CTR did not bind strongly to H3 (21-14) phospho-S28 
peptides, in agreement with both peptide array and beads pull- 
down analysis. 

We conclude that in vitro, the Topo Ha CTR binds directly 
to histone H3 and that modified isoforms of the H3 tail either 
enhance or inhibit binding. Because the Topo lla CTR can bind 

47B JCB • VOLUME SD3 • NUMBER 3 • 2D1 3 

to DNA and histone H3 at least in part through independent 
mechanisms, we refer to the last 31 residues of Topo Hot as the 
ChT domain. 

Topo 1 1 < v and histone H3 phospho-Ser 
SB are localized differentially on 
mitotic chromosomes 

If Topo Ila binds preferentially to certain histone H3 isoforms 
in vivo, then one possibility is that the localization patterns of 
such isoforms and of Topo Ila are related. Of particular inter- 
est is that phosphorylation of Ser 28 is associated with mitotic 
chromosomes (Goto et al., 2002), and it was therefore coun- 
terintuitive that this modification inhibits the interaction of H3 
and Topo Ila CTR in vitro (Fig. 4). To investigate this we immuno- 
stained the large mitotic chromosomes of M. muntjak using 
antibodies that have specificity for K27mel, K27me3, or S28p. 
Both K27 methylation-specific antibodies stained the entire width 
of mitotic chromosomes, with K27mel localizing with a more 
punctate pattern than K27me3 (Fig. 5, A and B). In contrast, 
S28p antibodies stained the periphery of chromosomes intensely, 
with weak staining within the perimeter of each chromosome. 
To ensure that this pattern was not caused by poor penetra- 
tion of antibody into chromosomes, we costained with antibod- 
ies against K27me3 and S28p. This revealed a clear contrast in 
localization patterns, where S28p was peripheral and K27me3 
was present throughout the width of chromosome arms (Fig. 5, 
C and D). 

We next asked how H3 isoforms localize relative to Topo 
Ila. Both K27 methylated isoforms localized with a pattern par- 
tially overlapping with the axial core localization of Topo Ila 
(Fig. 6, A and B), but S28p was clearly excluded from the cen- 
trally localized axial core (Fig. 6 C and Video 3). These immuno- 
localization studies of H3 isoforms are thus consistent with the 
biochemical analysis of binding between H3 isoforms and the 
Topo Ila CTR in vitro. 

A novel knockdown/rescue system for 
analysis of Topo Met mutants 

The data described so far indicate that the CTR, and in particu- 
lar the ChT domain, contains residues important for the asso- 
ciation of Topo Ila with mitotic chromosomes, at least in part 
through direct binding to DNA and histone H3. Because these 
interactions suggest a previously unknown mechanism of Topo Ila 
association with chromatin, we sought to test the functional 
relevance of the ChT domain in human cells. This was impor- 
tant for a further reason: In Saccharomyces cerevisiae, mutants 
lacking the CTR are viable (Jensen et al., 1996). A complication in 
addressing this question is that functional analysis of Topo Ila 
mutants in human cultured cells has previously been technically 
challenging. Transient transfection typically results in overex- 
pression of Topo Ila, leading to cell death. Carpenter and Porter 
(2004) previously generated a doxycycline-repressible Topo Ila 
human cell line and described in detail the consequences of 
loss of Topo Ila on chromosome condensation and segrega- 
tion. However, this system did not permit controlled expression 
of Topo Ila rescue constructs. Here, we developed a system in 
which Topo Ila (and mutants) are inducibly expressed close to 

endogenous levels and the endogenous Topo Ila can be effi- 
ciently depleted. 

The system employs a HeLa cell line (EM2-1 lht) in which 
a gene of interest is integrated stably at the well-characterized 
5q3 1 .3 "silent-but-activatable" genomic locus using the FLP 
recombinase (Fig. 7 A, left; Weidenfeld et al., 2009). This avoids 
detectable expression in the absence of doxycycline (Fig. 7 A, 
right). Constructs were inserted encoding WT mCherry-Topo Ila, 
mCherry-Topo Ila AChT, or mCherry-Topo Ila Y804F (a cata- 
lytic mutant of the active site tyrosine, serving as a negative 
control), and clonal lines were isolated. Because the locus of 
integration was the same for all cell lines generated, the induc- 
tion characteristics were similar, permitting a direct compari- 
son of independently derived transgenic cell lines. This enabled 
us to determine a concentration of doxycycline at which the 
level of transgenic mCherry-Topo Ila was similar to that of 
the endogenous protein (Fig. 7 A, right). Lentivirus-mediated 
shRNA resulted in depletion of endogenous Topo Ila to levels 
undetectable by immunoblotting (Fig. 7 A, right). Transgenic 
mCherry-Topo Ila and mutants of interest were rendered insen- 
sitive to the shRNA construct by introducing silent mutations 
before integration. 

AChT domain mutants have altered 
dynamics on mitotic chromosomes 

We first compared the localization pattern of mCherry-Topo Ila 
and mCherry-Topo Ha AChT. Although AChT clearly localized 
to interphase nuclei (Fig. S2) and to mitotic chromosomes in 
live cells (Fig. 7 B, top), it was entirely lost from chromosomes 
after fixation in formaldehyde or methanol (Fig. 7 B, bottom), 
whereas the full-length mCherry-Topo Ila was fully retained at 
centromeres and, to a lesser extent, at the axial core. Thus, topoi- 
somerases lacking the ChT domain must associate with chro- 
matin in a manner that becomes labile upon fixation. This result 
was the same whether or not the endogenous Topo Ila protein 
was depleted using shRNA (unpublished data). This indicates 
that the ChT domain is necessary for a stable association with 
chromosomes even in the presence of endogenous Topo Ila, 
where the mutant is presumably able to form a heterodimer. 

To study this phenomenon using a quantifiable approach 
in live cells, we used FRAP analysis. Previous work studying 
the dynamics of EGFP-Topo Ila used cells that overexpress 
the protein by an estimated several-fold (Tavormina et al., 2002), 
which may affect the apparent recovery dynamics due to, e.g., 
saturation of chromosomal binding sites. In addition to expres- 
sion at near-endogenous levels, our strategy used the mCherry 
fluorescent protein, which is more likely to remain monomeric 
at high concentrations of Topo Ila seen on mitotic chromosomes 
than the weakly dimeric EGFP (Zacharias et al., 2002). 

We found that the mCherry-tagged WT protein is highly 
mobile on chromatin in metaphase cells, with a recovery t m of 
10.3 s (Fig. 8, A and B). This is slightly faster than the published 
data that yielded a t m of 15.5 s (Tavormina et al., 2002). Impor- 
tantly, however, we observed that mCherry-Topo Ila AChT had 
an increased mobility such that the recovery t m was reduced to 
6.4 s. This is consistent with a higher Ko ff than the WT protein 
and the loss of chromatin interactions when compared with the 

Mechanism of topoisomerase II chromatin localization and dynamics • Lane et al. A~7~7 

Figure 5. Immunolocalization of histone H3 
isoforms in mitotic M. muntjak chromosomes. 

M. muntjak cells stained with antibodies 
against H3K27mel (A] or H3K27me3 (B), 
or costained with H3K27me3 and H3S28p 
(Cand D). H3K27mel and H3K27me3 aredis- 
tributed throughout chromosome arms, whereas 
H3S28p is more peripheral. Flattened z stacks 
are shown. DAPI, DNA stain. Bars, 10 urn. 
Images on the right show enlarged segments 
of the boxed regions (bars, 1 .25 urn). 

^^K27me3 /H3 


H3S28p /H3K27me3,S; 



WT protein. Again, these experiments were performed without 
depletion of the endogenous Topo Ila, which indicates that the 
difference in mobility is evident even when there presumably 
exists a population of WT/mutant heterodimers. For this reason, 
our estimate of the reduction in residence time of Topo Ila lacking 

the ChT domain is likely to be conservative. Moreover, the data 
suggest that in WT cells, two intact ChT domains are required for 
the stable association of the Topo Ila homodimer with chromatin. 

In sum, the interaction of mCherry-Topo Ila AChT is 
more labile upon fixation of cells, and in live mitotic cells it is 

JCB • VOLUME SD3 • NUMBER 3 • 2D1 3 

H3K27me3/Topo I la 

H3S28p/Topo Ma 

Topo I la 

bit ^^^^| 

Figure 6. Immunolocalization of histone H3 isoforms and Topo lla in mitotic M. muntjak chromosomes. M. muntjak cells stained with anti-GFP antibody to 
recognize GFP-Topo lla and costained with antibodies against H3K27mel (A), H3K27me3 (B), or H3S28p (C). H3K27mel and H3K27me3 are distributed 
throughout chromosome arms and partially colocalize with Topo lla. H3S28p localizes peripherally to Topo lla. Flattened z stacks are shown. Bar, 1 0 urn. 
The full z-series of C is shown in Video 3. DAPI, DNA stain. Images on the right show enlarged segments of the boxed regions (bars, 1 .25 urn). 

more mobile, which is consistent with weaker affinity inter- 
actions with chromatin. The ChT domain dictates the dynamics 
of Topo Hot on mitotic chromosomes. 

AChT domain mutants cannot facilitate 
chromosome individualization, resolution, 
or condensation 

We next investigated the biological consequences of the altered 
dynamics of mCherry-Topo lla AChT in terms of mitotic chro- 
mosome formation. Because previous studies have shown that 
Topo Hp can weakly compensate for the lack of Topo Ilct 
(Linka et al., 2007), we codepleted both isoforms, using shRNA 
lentiviral transduction, to achieve a minimum base level of type II 
topoisomerase activity (Fig. S3). We simultaneously induced 
expression of mCherry-Topo Hot, mCherry-Topo lla AChT, or 
mCherry-Topo lla Y804F (as a negative control) in the isogenic 
cell lines and monitored changes in metaphase chromosome 

morphology when cells reached mitosis after release from a 
double thymidine synchrony. Giemsa staining of Carnoy's fixed 
cells allowed fine-scale examination of changes in chromosome 
morphology (Gimenez-Abian et al., 1995; Gimenez-Abian and 
Clarke, 2009; Fig. 8, C and D). In addition, we filmed cells, 
treated in the same manner, by digital time-lapse microscopy to 
observe single live cells as they entered and progressed through 
mitosis (Fig. 9). 

In cells depleted of type II topoisomerases, we saw large 
numbers of mitotic cells with chromosomes that failed to become 
individualized (Fig. 8 C and Fig. 8 D, right). This is identical to 
the morphology observed when cells attempt to condense their 
chromosomes in the presence of high doses of Topo lla catalytic 
inhibitors (Gimenez-Abian and Clarke, 2009). Less frequently, 
we observed undercondensed chromosomes in which the sis- 
ter chromatids had failed to become resolved into discrete pairs 
(Fig. 8 C and Fig. 8 D, middle). This is identical to the results of 

Mechanism of topoisomerase II chromatin localization and dynamics • Lane et al. 479 

Figure 7. An inducible system for analysis 
of Topo lla mutants. (A, left) Schematic de- 
scribing the strategy to derive HeLa EM2-1 1 ht 
cells expressing RNAi-resistant mCherry- 
Topo lla at close to endogenous levels. Single 
copy insertion of Topo lla alleles at locus 
5q31.3 was achieved using asymmetric FLP 
(F/F3) recombinase sites (see Materials and 
methods and Weidenfeld etal., 2009]. (A, right) 
Immunoblot detecting endogenous Topo lla and 
exogenously expressed mCherry-Topo lla. 
HeLa EM2-1 1 cells were infected with lentivi- 
ral particles encoding knockdown shRNA con- 
structs at day 0 against Topo lla and Topo Hp 
simultaneously with the addition of 250 ng/ml 
doxycycline to induce expression of the ex- 
ogenous allele. Endogenous Topo lla is ef- 
fectively depleted, whereas the exogenous 
protein is expressed to approximately endog- 
enous levels within 24 h. (B) Localization of 
mCherry-Topo lla and mCherry-Topo lla 
AChT after 48 h of induction with doxycycline 
in live HeLa EM2-1 1 cells (top) or after fixa- 
tion with methanol (bottom). Topo lla AChT is 
labile upon fixation. Bars, 10 pm. 

FLP-recombinase mediated gene exchange 

pTet:mCherry-Topo lla RNAi R 

Added Dox and Topo lla 
shRNA vims 

MW (ku) ▼ 
Anti Topo Ma 200 ' 

. -4 mCherry-Topo lla 
Endogenous Topo No 

Live - 

Topo lla 

Topo lla AChT 


Topo lla 

Topo lla AChT 

Anti aTubulin 50 . 

Day 0 1 2 3 4 

mCherry DNA 

mCherry DNA 



treatment with lower doses of Topo II catalytic inhibitors that 
restrict but do not eliminate Topo II activity (Gimenez-Abian 
and Clarke, 2009). Control cells treated with a nontargeted 
shRNA virus showed normal mitotic chromosome morpholo- 
gies with well-individualized and condensed chromosomes that 
had fully resolved sister chromatids (Fig. 8 D, left). The time- 
lapse analysis of single cells was consistent with these data and 
in addition revealed obvious defects in chromosome segregation 
and progression through mitosis (Fig. 9 and Videos 4-9). 

Strikingly, the induction of mCherry-tagged Topo Hot 
rescued the condensation-, individualization-, and resolution- 
defective phenotypes in >75% of the fixed cells analyzed 
(Fig. 8 C). Similarly, based on the analysis of single live cells, 
the mitotic defects were rescued in ^60% of the cells (Fig. 9 C). 
Thus, the mCherry-tagged protein can functionally complement 
the loss of endogenous Topo Ilct, and the shRNA vectors did 
not have significant off-target effects that affected chromosome 
morphology. However, neither the induction of mCherry-Topo 
Hot AChT or of mCherry-Topo lla Y804F was able to rescue 
the chromosome morphology defects, which indicates that they 
are each deficient in the formation of mitotic chromosomes 
(Fig. 8 C and Fig. 9 C). This is the first indication that the pre- 
cise dynamics of Topo Ilct on mitotic chromosomes may be 
required for the chromosome structural changes needed for suc- 
cessful mitosis in mammalian cells. 


Our data demonstrate that a novel ChT domain at the extreme 
C terminus of Topo lla increases the residency time on mitotic 
chromosomes in a manner that correlates with histone H3 and 
DNA binding in vitro and which is essential for chromosome 
condensation. This suggests that the precise dynamics of Topo Hot 
are crucial for assembly of mitotic chromosomes. Insight into 
the interaction between Topo lla and chromatin is significant 
for ongoing research efforts to maximize the efficacy of Topo II- 
targeted cancer drags because tumor cells with reduced strand 
passage activity become drug resistant (Nitiss, 2009), and one 
such route to loss of activity is through mutations that reduce 
chromatin binding. 

The ChT domain is required for a full-affinity interaction 
with both DNA and histone H3 in vitro and with mitotic chro- 
mosomes in the context of full-length Topo Hot. It possesses 
an N-terminal stretch of nonpolar residues interspersed with 
Arg and Lys, and a C-terminal stretch of acidic amino acids 
(Fig. 2 A). Because the ChT domain is implicated in an inter- 
action with both DNA and histone H3, a simple predication is 
that the acidic stretch contacts the basic H3 N-terminal tail and 
that the DNA associated with or adjacent to the correspond- 
ing nucleosome interacts with the Lys and Arg residues of the 
ChT domain. In agreement with this possibility, we observed 

JCB • VOLUME SD3 • NUMBER 3 • 2D1 3 



| o 


& 0 


t 0 


8 - 






I 1 : 

" Topo I la 
* Topo lla AChT 
ti/sWT: 10.3s 
ti/2AChT: 6.4s 

Distribution of mitotic chromosome phenotypes in 
HeLa cells expressing TOPOIIa WT, AChT or 
catalysis-dead (Y804F) mutant 

-5 0 

I 1 1 1 r 

10 20 30 40 50 

Time post-bleach (s) 



resolution defect 

i i i 

25% 50% 75% 
Percent phenotype 

resolution defect 


Figure 8. The ChT domain controls the dyna- 
mics of Topo lla on mitotic chromosomes and 
is necessary for mitotic chromosome individu- 
alization and condensation. (A) Images of a 
metaphase plate from a representative FRAP 
experiment using HeLa-EM2-l 1 mCherry- 
Topo lla cells 48 h after induction with doxy- 
cycline. A laser-scanning confocal microscope 
was used to bleach a circular region (broken 
line) encompassing ~50% of the metaphase 
plate, and recovery was monitored over time 
(seconds). Pre, prebleach image. Bar, 10 pm. 

(B) Mean FRAP of mCherry-Topo lla (n = 1 1 
cells) and mCherry-Topo lla AChT [n = 2 1 ) in 
HeLa-EM2-l 1 cells, as described in A. After 
collection of prebleach data (top left of plot), 
images were collected every 0.43 s. Recovery 
was quantified in the bleached area over 50 s 
and normalized as described previously for 
analysis of chromatin proteins (Phair et al., 
2004a). Error bars indicate standard error. 

(C) Distribution of phenotypes seen in cells 
depleted of endogenous Topo II and induced 
to express mCherry-Topo lla, mCherry-Topo lla 
AChT, or mCherry-Topo lla Y804F . Induction 
of mCherry-Topo lla rescues chromosome 
condensation, individualization, and resolu- 
tion defects, whereas Topo lla AChT and 
Topo lla Y804F fail to rescue. Cells were assayed 
after a double thymidine synchrony and re- 
lease protocol in conjunction with shRNA- 
mediated depletion of the endogenous Topo II 
(see Materials and methods). WT — Dox, 
n = 129; WT + Dox, n = 141; AChT - Dox, 
n = 100; AChT + Dox, n = 100; Y804F - 
Dox, n = 148; Y804F + Dox, n = 146. 

(D) Classification used in C. Spreads of chro- 
mosomes after depletion of endogenous Topo 
II show a range of mitotic phenotypes: "Nor- 
mal" metaphase plates containing individual- 
ized chromosomes with visibly resolved sister 
chromatids (left, red in C), chromosomes that 
failed to complete linear condensation and sis- 
ter chromatid resolution (middle, green in C), 
and failed chromosome individualization and 
sister chromatid resolution (right, blue in C). 
Bars, 10 pm. Insets show enlarged views of 
the boxed regions (bars, 1 .25 pm). 

that the acidic residues are required for the in vitro interaction 
with H3. However, because a crystal structure including the 
entire CTR of human Topo lla does not exist, we are limited 
to speculating on its likely tertiary structure. Moreover, our 
data indicate that the interaction surfaces between chromatin 
and the CTR are still more complex than the residues within 
the ChT domain that we have shown to be crucial. First, the 
CTR lacking the ChT domain retains 60% of the binding ca- 
pacity for DNA in vitro, indicating that other CTR residues 
contact DNA. Second, we observed that the Topo Hot K3R 
mutant has slightly reduced affinity for histone H3 in vitro 
(Fig. S4). A structural approach will be needed to determine 
all of the contact residues between DNA, histone H3, and the 
Topo lla CTR. 

The K3R mutant studies revealed that Topo Hot does not 
need to be in the nucleus during interphase in order for it to 
localize to mitotic chromosomes in mitosis (Fig. 2). However, 

in support of the biological importance of the K3 residues, Topo 
lla K3R had altered dynamics on mitotic chromosomes and was 
defective for chromosome formation in mitosis in the absence 
of the endogenous Topo Hot (Fig. S4). We cannot distinguish 
whether these defects arise as a consequence of the lack of Topo 
Ha K3R in the nucleus during DNA replication, which is con- 
sistent with previous studies (Cuvier and Hirano, 2003), or are 
due to reduced affinity for histone H3. 

We also provided evidence that the precise dynamics of 
Topo Hot, dictated by the ChT domain, are important for chro- 
mosome individualization and condensation, and for sister chro- 
matid resolution in mitosis. This indicates that the dissociation 
frequency of the enzyme from chromatin may dictate the abil- 
ity of the enzyme to perform its mitotic functions. The AChT 
mutant had about a 40% decrease in recovery t l/2 versus WT 
Topo Hot. However, this is likely to be a conservative estimate 
of the defect observed because the FRAP studies were performed 

Mechanism of topoisomerase II chromatin localization and dynamics • Lane et al. 



















t— x 


O Q 

< + 

-c o 
O Q 





















- w 















































5:30 11:00 

WT +Dox 



AChT +Dox 


AChT -Dox 


WT + Dox 


AChT + Dox 

AChT -Dox 

l l Normal 

' — ' condensation 

| Failed 






Figure 9. The ChT domain of Topo Met is necessary for chromosome condensation and segregation in mitosis. (A and B) Still images taken from digital time- 
lapse imaging of live cells after depletion of endogenous Topo Ha and induced with doxycycline to express mCherry-Topo Ha or mCherry-Topo Ha AChT. 
Cells were assayed after a double thymidine synchrony and release protocol in conjunction with shRNA-mediated depletion of the endogenous Topo lla 
and DNA observed via imaging of Hoechst. Red boxed regions are enlarged in B to highlight the differences in metaphase chromatin morphology. Frames 
were taken at 5-min intervals. Time is indicated (hours:minutes) in each movie frame. Bars, 10 urn. (C) Distribution of phenotypes seen in live single cells 
described in A and B. Induction of mCherry-Topo lla rescues chromosome condensation and segregation defects, whereas Topo lla AChT fails to rescue. 
WT - Dox, n = 96; WT + Dox, n = 1 00; AChT - Dox, n = 61; AChT + Dox, n = 82. 

in the presence of the endogenous WT protein, which poten- 
tially allowed the formation of Topo lla AChT/Topo lla het- 
erodimers with an intermediate affinity for chromatin. Recent 
work led to the estimate that Topo Hot performs around 10 en- 
zyme cycles before releasing from chromatin (Yogo et al., 2012). 
The increased dissociation rate of the Topo Hot AChT mutant, as 
measured by FRAP, presumably decreases the processivity of 
the enzyme, most likely via the loss of interactions with histone 
H3 and DNA. It will be of interest to determine how binding of 
the ChT domain to chromatin is coordinated in the context of the 
enzyme cycle: whether the ChT domains of the homodimeric 

enzyme associate with both G-segment and T-segment chroma- 
tin or specifically with just one of the segments, and if the inter- 
actions are lost during any of the large conformational changes 
through which the enzyme transits during strand passage. In- 
triguingly, the acidic stretch of residues essential for histone H3 
binding contains a nested serine (Ser 1524), which, upon 
phosphorylation stimulated by Topo lla catalytic inhibitors, in- 
teracts with the cell cycle checkpoint protein MDC1 (Luo et al., 
2009). Presumably, there must be a competition in vivo between 
histone H3 and MDC1 for ChT domain occupancy. If H3 binding 
is intimately linked with strand passage, then interruption of the 

4B2 JCB • VOLUME 2D3 • NUMBER 3 • 2D1 3 

reaction cycle may lead to release of H3, allowing phosphoryla- 
tion of Ser 1524 and checkpoint activation via MDC1 binding. 
Why does Topo Hot interact with histone H3? Estimates of a 
mean internucleosomal linker DNA length of ^54 bp (Schones 
et al., 2008), of which Topo Ila contacts 26 bp in coordinating a 
double-strand break, suggest that highly nucleosome-occupied 
areas of chromatin may present relatively limited opportunities 
for Topo Ila activity. Indeed, it has been reported that Topo Ila 
activity is inhibited on nucleosome-occupied DNA in vitro 
(Galande and Muniyappa, 1997). An attractive hypothesis is 
that the ChT domain serves to mitigate the inhibitory effect of 
nucleosomes, perhaps by positioning the enzyme adjacent to a 
histone octamer, or more simply, increasing the residence time 
of the enzyme at nucleosome-rich regions such that its rate of 
catalytically productive engagement is raised nearby. 

Immunostaining of M. muntjak chromosomes revealed 
strikingly different localization patterns of H3 isoforms in mitotic 
chromosomes. The methylated K27 isoforms partially over- 
lapped with Topo Ila within the axial core (Figs. 5 and 6). 
Similarly, superresolution microscopy studies have shown that 
the K27mel isoform is partly confined to the interior of Dro- 
sophila mitotic chromosomes (Strukov et al., 2011). Most inter- 
estingly, we observed that H3 phosphor-Ser 28 is largely excluded 
from the core region of M. muntjak chromosomes. The Aurora B 
kinase that modifies H3 Ser 28 has multiple substrates. Never- 
theless, it is intriguing that inhibition of Aurora B alters the 
localization of Topo Ila on mitotic chromosomes (Fig. S5). 
Together, these studies raise the possibility that the status of 
histone tail modifications controls the association of Topo Ila 
with chromatin. It will be of interest to determine if other chro- 
mosomal proteins possess ChT domains and whether they bind 
to particular histone isoforms. 

Materials and methods 


The pTl 04-1 EGFP-Topo Ila expression vector contains a Sacll-Hindlll frag- 
ment encoding human Topo Ila cloned in-frame into pEGFP-C3 under a 
cytomegalovirus promoter (Takara Bio Inc.; Mo et al., 1998). Site-directed 
mutagenesis to generate the K3R, Y804F, and truncation mutants used the 
method of Liu and Naismith (2008). mCherry-tagged CTR constructs were 
constructed using pmCherry-Cl (Takara Bio Inc.), and PCR-amplified prod- 
ucts encoding C-terminal fragments from pT 1 04- 1 (or mutants) were cloned in 
at Bglll-Sacll; the resultant constructs are under a cytomegalovirus promoter. 
S2F-IMCg-F-EGFP/luc-F3 vectors were a gift from K. Schoenig (Zentralinstitut 
fur Seelische Gesundheit [Zl], Mannheim, Germany; Weidenfeld et al., 
2009). To generate FLP-in Topo Ila vectors, EGFP was removed from the 
self-inactivating viral vector S2F-IMCg-F-EGFP/luc-F3 using Sacll-Notl sites 
and replaced with an mCherry PCR product containing an additional 3' 
Mlul site to render S2F-IMCg-F-mCherry/luc-F3. Topo Ila was amplified by 
PCR to contain Mlul sites at both ends and cloned into S2F-IMCg-mCherry/ 
luc-F3 to render S2F-IMCg-F-mCherry-Topo lla/luc-F3; expression is thus 
under an rtTA (Tet-On) promoter. All growth of S2F-IMCg vectors was per- 
formed at 30°C to reduce the frequency of plasmid rearrangement. Con- 
structs used for expression of Topo lla/p fragments in Escherichia coli were 
generated using PCR-amplified fragments from pT 1 04-1 or Topo IIB 1 1 86- 
1621 (Linka et al., 2007) cloned into pET28a (EMD Millipore) at Sacl- 
Hindlll under a T7/lac promoter. Constructs encoding Topo Ila with silent 
mutations conferring resistance to shRNA constructs V3LHS_327878 and 
V3LHS_327876 were constructed as follows. Fragments of pT 1 04- 1 were 
amplified using primers containing a 5' synthetic shRNA-resistant sequence 
and a 3' adjoining Topo Ila sequence. Vector IMCg-F-mCherry-Topo Ila/ 
luc-F3 was amplified using similar primers, designed to amplify around the 
plasmid. The resulting PCR products thus contained homology in the ~20 bp 

shRNA-resistance regions at the ends of each product and could be combined 
using sequence and ligation-independent (SLIC) cloning (Li and Elledge, 
2007). shRNA-resistant regions were then subcloned out of the resulting 
vector and cloned back into IMCg-F-mCherry-Topo lla/luc-F3 by traditional 
(restriction enzyme-mediated) methods to avoid mutations generated dur- 
ing vector PCR. This process generated IMCg-F-mCherry-Topo lla/luc-F3 
shRNA R (thus all Topo Ila constructs also express luciferase from a coregu- 
lated promoter). 

FLP-in cell line generation 

HeLa EM2-1 1 ht cells were a gift from K. Schoenig. Recombination-mediated 
cassette exchange was achieved using cotransfection of S2f-IMCg-Topo Ila/ 
luc-F3 and pPGKFLPobpA (Flp recombinase encoding plasmid; Raymond 
and Soriano, 2007), and Addgene plasmid 13793 and pTRE2-pur 
(Takara Bio Inc.) in a 6-well dish. At t = 24 h, cells were trypsinized and 
plated in 4 pg/ml puromycin. At t = 48 h, existing media was replaced 
with media containing 50-100 \iM ganciclovir. Cells were cultured with 
ganciclovir for 10-14 d until colonies were visible, with media changes 
every 48 h . 

Topo Ila shRNA knockdowns 

Knockdowns used GIPZ clones Topo Ila V3LHS_327878, Topo lip V2LHS_ 
94084, or accompanying GIPZ nonsilencing control sequence vector (cat- 
alog no. RHS4346; Thermo Fisher Scientific). Viruses were produced by 
cotransfection of HEK293T cells with GIPZ vector and pMDG and ANRF 
packaging constructs using linear polyethylenimine (PEI) at a 3:1 DNA/PEI 
ratio. Viral transductions were performed using standard protocols in the 
presence of 8 ug/ml Polybrene. 

DNA transfections 

Transfections were performed using Genjet Plus reagent (SignaGen Labo- 
ratories) according to the manufacturer's instructions. 


Mitotic cells were obtained by shake-off, then allowed to adhere onto 
poly-L-lysine-coated coverslips before fixation with 4% paraformalde- 
hyde for 1 0 min, post-fix extraction with 0.5% Triton X-l 00 for 5 min, and 
further fixation in — 20°C methanol for 10 min. Blocking was achieved 
in wash buffer (PBS with 0.01% Triton X-l 00) with 3% BSA (Sigma- 
Aldrich). Antibodies used were: a-H3S28phos (catalogue no. H9908; 
Sigma-Aldrich), a-H3K27mel (catalogue no. A-4037; Epigentek), a- 
H3K27me3 (catalogue no. 07-449; EMD Millipore), a-GFP (catalogue 
no. 1814460; Roche), goata-ratCy3 (catalogue no. 1 1 2-1 65-1 43; Jackson 
ImmunoResearch Labs), goat a-rabbit IgG Alexa Fluor 488 (catalogue 
no. Al 1008; Molecular Probes; Invitrogen), goat a-mouse IgG 1 Alexa 
Fluor 488 (catalogue no. A21121; Molecular Probes, Invitrogen), and 
goat a-rabbit IgG (H+L) Alexa Fluor 568 (catalogue no. Al 1 01 1 ; Molecular 
Probes, Invitrogen). 

Fluorescence and bright field microscopy 

Fluorescence imaging was performed using a DeltaVision PersonalDV 
microscope system (Applied Precision), a 1 OOx Uplan S Apochromat (NA 
1 .4) objective lens (Olympus), and a CoolSNAP HQ2 camera (Photomet- 
ries). Fixed material was imaged at ambient temperature after mounting 
in Vectashield (Vector Laboratories). Live cell time-lapse analysis was con- 
ducted using the DeltaVision weather station chamber at 37°C in C0 2 - 
independent medium, and images were collected at 5-min intervals. Images 
were acquired using DeltaVision SoftWoRx software (Applied Precision) 
and processed using ImageJ (Abrdmoff et al., 2004). If necessary, cam- 
era hot pixels removed using Remove Outliers; care was taken to avoid 
altering chromosome signal. Line profiles in Figs. 1-3 were calculated 
using the Plot Profile tool in ImageJ with a line width of 20 pixels and plot- 
ted relative to background (nonchromosomal) areas. Where indicated, 
z stacks were deconvolved using DeltaVision SoftWoRx software (Applied 
Precision). Fluorochromes used for immunostaining were as follows: Cy3 
(goat a-rat conjugate, catalogue no. 112-165-143; Jackson Immuno- 
Research Laboratories, Inc.), Alexa Fluor 488 (goat a-rabbit IgG conjugated, 
catalogue no. Al 1008; Molecular Probes; Invitrogen), Alexa Fluor 488 
(goat a-mouse IgG 1 conjugate, catalogue, no. A21121; Molecular 
Probes; Invitrogen), Alexa Fluor 568 (goat a-rabbit IgG [H+L] conjugate; 
catalogue no. A 1 101 1 ; Molecular Probes; Invitrogen). Bright-field mi- 
croscopy: Giemsa-stained chromosomes (mounted in Entellan; EM Sci- 
ence) were imaged using an Axioplan2 microscope (Carl Zeiss), a lOOx 
a-Plan Fluor (NA 1 .45) objective lens, and an AxioCam HRm camera 
(Carl Zeiss) at ambient temperature. 

Mechanism of topoisomerase II chromatin localization and dynamics • Lane et al. 4B3 

Cytological analysis of chromosome structure 

shRNA-transduced cells were synchronized using the double-thymidine ar- 
rest protocol. In brief, beginning 52 h after shRNA transduction/doxycycline 
addition, cells were incubated for 16 h in 2 mM thymidine. Thymidine was 
then washed out and the cells cultured for 1 0 h. The culture medium was then 
resupplemented with 2 mM thymidine for 16 h. Thymidine was washed out 
a second time and cells were harvested 10 h later in M phase. Chromosome 
preparations were prepared by osmotic swelling in 50% medium/50% tap 
water for 7 min, followed by three washed in Carnoy's fixative (75% meth- 
anol, 25% glacial acetic acid; Gimenez-Abidn and Clarke, 2009). After 
dropping the material onto cleaned glass slides, chromosomes were stained 
5% Giemsa (EMD) in phosphate buffer, pH 6.8 (Harleco; EMD). 

Protein expression and purification 

Fragments of human Topo Ha or Topo 11(3 were amplified from pT 1 04- 1 or 
from YFP-Topo 11(3 expression plasmids as used in Linka et al. (2007), 
cloned into pET28a, and transformed into BL21 (DE3). Expression was 
induced using the autoinduction method (Studier, 2005) in rich media, 
cells were lysed using lysozyme and sonication, and the supernatant was 
purified on Ni-NTA resin (QIAGEN) under nondenaturing conditions. Pro- 
teins were quantified using a BCA assay (Thermo Fisher Scientific). 

Electromobility shift assays 

Purified recombinant Topo Met fragments were incubated with purified 
pUC 1 9 DNA in bandshift buffer (Campoy et al., 1 995), prepared without 
E. coli competitor DNA, for 30 min at RT. Samples were loaded onto 1% 
agarose gels in 0.5x Tris/Borate/EDTA (TBE) and run at 1 .2 V/cm at4°C 
for 20 h, then post-stained with ethidium bromide, based on the protocol 
of Yang and Champoux (2009). 

DNA binding assays by pull-down 

Assays were performed using a modified version of Wu (2006), as follows. 
Complimentary 60-bp 5' biotin-conjugated primers (Integrated DNA Tech- 
nologies) were annealed by mixing in an equimolar ratio, heating to 1 00°C, 
and cooling to RT. The resulting double-stranded DNA (dsDNA) was incu- 
bated with Streptavidin-coated Magnabeads (Genscript) for 30 min at RT, 
rotating end over end. The resulting oligonucleotide-coated beads were then 
blocked with 1 0% milk (filtered to remove particles) and 1 % NP-40 in PBS 
for 50 min at RT. Beads were washed once in PBS/1% NP-40/ 1% milk, in- 
cubated with purified recombinant Topo Ha or Topo 11(3 fragments in the same 
solution for 3 h at RT, washed twice in PBS/1 % NP-40, once in PBS, and then 
boiled in SDS sample buffer and resolved on a 1 5% SDS-PAGE gel. The protein 
was transferred to polyvinylidene fluoride (PVDF) and subjected to immuno- 
detection using anti-His primary antibody HI 029 (Sigma-Aldrich) and anti- 
mouse secondary antibody conjugated to IR680 dye and scanned using a 
LI-COR Odyssey blot scanner (LI-COR Biosciences). 

Western blotting and protein extraction 

HeLa cells were harvested and lysed in TBS/2% SDS, 1 x Complete prote- 
ase inhibitor (Roche), and sonicated to shear DNA. Protein was quantified 
using a BCA protein assay kit (Thermo Fisher Scientific) and loaded onto 
SDS-PAGE gels, run as standard. Protein was transferred to PVDF and 
probed using anti-Topo lla antibody sc-13058 (Santa Cruz Biotechnol- 
ogy, Inc.) or anti-a-Tubulin antibody abl5246 (Abeam). 

Modified histone arrays 

Histone peptide arrays (Active Motif) were blocked using 5% nonfat dried 
milk and 1 % porcine gelatin for 2 h. Arrays were then incubated with 200 nM 
HIS-Topo lla CTR for 1 h. Bound Topo lla was detected using anti-HIS 
monoclonal antibody at 1 :5,000 (H 1 029; Sigma-Aldrich) and HRP-conjugated 
anti-mouse secondary antibody at 1 :3,000 (Invitrogen) for chemiluminescent 
detection. All solutions were prepared in TBS containing 0.02% Tween 20, 
and washes in this buffer were performed between steps. 

Topo lla fragment pull-downs from HeLa cell lysates 

HeLa cells were lysed using NP-40 buffer (20 mM Tris, pH 8, 1 37 mM 
NaCI, 10% glycerol, 1% NP-40, 2 mM EDTA, lx Complete protease in- 
hibitors [Roche], 420 ug/ml NaF, 1 .84 mg/ml Na 3 V0 4 , 10 mM N-ethyl- 
maleimide, 5 U/ml cynase [Ribosolutions, Inc.], and 4 mMMgCb). Lysates 
were incubated with anti-HIS Magnabeads at 4°C overnight, then washed 
in PBS/0. 1 % NP-40. Bound proteins were eluted in SDS-PAGE loading buf- 
fer and analyzed by SDS-PAGE and silver staining (Bio-Rad Laboratories). 

In vitro pull-down assays 

To study interactions between Topo lla CTR and biotinylated H3 peptides 
(residues 21-44), 25 pi of Streptavidin Dynabeads MyOne Tl were 

washed in binding buffer (50 mM NaH 2 P0 4 , pH 7.5, 50 mM NaCI, 
0.05% NP-40, and 10 mg/ml BSA) three times before resuspending in a 
complex of 3 pg peptide and 10 pg purified HIS-Topo lla CTR that had 
been incubated overnight at 4°C in a volume of 50 pi. Peptides/Topo lla 
CTR complexes were conjugated to beads in a 2 h, 30 min incubation at 
RT. Beads were washed three times in 150 pi of binding buffer without 
BSA. For analysis, beads were boiled in 15 pi SDS-PAGE loading buffer 
and separated on 4-1 2% SDS-PAGE gels. 

To study interactions between HIS-Topo lla CTR and full-length 
H3, 2.5 pg of mouse anti-HIS antibody (Takara Bio Inc.) per sample was 
incubated with 15 pi Protein A Dynabeads in 25 pi of 50 mM NaH 2 P0 4 , 
pH 8, 0.05% NP-40, 50 mM NaCI, and 1 0 mg/ml BSA per sample over- 
night at 4°C. Beads were washed in 1 50 pi of the same buffer and resus- 
pended in 50 pi. Simultaneous to antibody-bead conjugation, HIS-Topo lla 
CTR (0.75 pg) and/or H3.1 or H3.3 (1.5 pg) as appropriate were in- 
cubated in a volume of 25 pi of 50 mM NaH 2 P0 4 , pH 7.5, 50 mM 
NaCI, 0.05% NP-40, and 10 mg/ml BSA overnight at 4°C. HIS-Topo 
lla CTR/H3 complexes were incubated with antibody-conjugated beads 
for 2 h and 30 min at RT. Beads were washed three times in 150 pi of 
50 mM NaH 2 P0 4 , pH 7.5, 50 mM NaCI, and 0.05% NP-40, then 
resuspended in 1 5 pi SDS-PAGE loading buffer and separated on 4-1 2% 
SDS-PAGE gels. 

ELISA assays 

96-well MaxiSorp plates (Thermo Fisher Scientific) were coated with 5 pg/ ml 
NeutrAvidin in 200 mM carbonate/bicarbonate buffer, pH 9.4, for 48 h 
at 4°C. Wells were blocked with 300 pi PBS containing 5 mg/ml BSA for 
45 min. Unmodified, methylated, or phosphorylated histone H3 (21-44) 
C-biotinylated peptides (AnaSpec) were resuspended to 100 ng/ml in PBS 
containing 10 mg/ml BSA and 0.05% Tween 20, and incubated (100 pi 
per well) for 1 h at RT. Wells were washed using 300 pi of wash buffer 
(PBS containing 0.05% Tween 20) three times before adding 100 pi HIS- 
Topo lla CTR (1321-1530) at various concentrations in 50 mM NaH 2 P0 4 , 
pH 7.5, 50 mM NaCI, 10 mg/ml BSA, and 0.05% NP-40 and incubating 
for 1 h at RT. After washing three times with 300 pi wash buffer, bound 
HIS-Topo lla CTR was detected using 1 :2,500 anti-HIS antibody (Takara 
Bio Inc.) in PBS containing 10 mg/ml BSA and 0.05% NP-40 for 1 h. After 
washing again, wells were incubated for 30 min with 1 :2,500 goat anti- 
mouse ECL (HRP-conjugated) secondary antibody (GE Healthcare). After 
an additional wash step, HRP signal was developed using TMB substrate 
(R&D Systems) according to the manufacturer's protocol. Plates were read 
at 450 nm on a SpectraMax M2 plate reader (Molecular Devices). After 
subtraction of negative control values to account for nonspecific binding, 
absorbance values were fitted to a four parameter logistic curve using the 
drc package in the R Statistical Computing environment. 


FRAP using HeLa EM2-1 1 ht cells was performed using a point-scanning con- 
focal microscope (FluoView 1000 1X2; Olympus), 100x/1.3 NA UPlanFL 
objective lens, and FluoView acquisition software. Cells were maintained 
in DMEM containing Hepes buffer on a Delta-T heated dish system (Bioptechs, 
Inc.) at 37°C. After data collection, images were analyzed using ImageJ, 
and intensity values were double-normalized as per Phair et al. (2004b) 
and scaled to between 0 and 1 . 

Online supplemental material 

Fig. SI shows Topo lla CTR binding to an expanded H3 tail peptide array. 
Fig. S2 shows localization of Topo lla AChT in interphase. Fig. S3 shows 
a Western blot of HeLa cells expressing exogenous Topo lla mutants and 
depleted of endogenous Topo lla. Fig. S4 shows analysis of Topo lla K3R 
mutant. Fig. S5 shows localization of Topo lla after Aurora B inhibition 
in mitosis. Video 1 shows a 3D reconstruction of Topo lla localization in 
M. muntjak cells. Video 2 shows Topo lla K3R localization in a live cell 
entering mitosis. Video 3 shows z sections through a fixed /VI. muntjak cell 
costained to localize Topo lla and H3S28p. Videos 4-9 show live HeLa 
cells depleted of Topo lla and induced to express either Topo lla or Topo lla 
AChT. Online supplemental material is available at 
cgi/content/full/jcb.201 303045/DC1 . Additional data are available in 
the JCB DataViewer at! 0. 1 083/jcb.201 303045.dv. 

We thank Ryoko Kuriyama, Melissa Gardner, Meg Titus, Dennis Livingston, 
Gant Luxton, and Rebecca Heald for insightful comments on the manuscript; 
Hung-Ji Tsai, Jeremy Wilbur, Ina Weidenfeld, Kai Schonig, and Andrew Grenfell 
for input on experimental procedures; and Mark Sanders, Guillermo Marques, 
and the University of Minnesota Biomedical image Processing Laboratory for 
assistance with the FRAP studies. 

4B4 JCB • VOLUME 203 • NUMBER 3 • 2D1 3 

This work was supported in part by National Institutes of Health grant 
CA099033, National Science Foundation grant MCB-0842 1 57 (DJC), and 
the Minnesota Medical Foundation (all to D.J. Clarke]. 

Submitted: 8 March 201 3 
Accepted: 8 October 201 3 


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4BB JCB • VOLUME 203 • NUMBER 3 • 201 3