The EMBO Journal Peer Review Process File - EMBO-2012-83176
Manuscript EMBO-2012-83176
Prophase Pathway-Dependent Removal of Cohesin from
Human Chromosomes Requires Opening of the Smc3-Scc1
Gate
Johannes Buheitel and Olaf Stemmann
Corresponding author: Johannes Buheitel, University of Bayreuth
Review timeline:
Submission date:
Editorial Decision:
Revision received:
Acceptance letter:
Accepted:
01 September 2012
03 October 2012
11 December 2012
08 January 2013
08 January 2013
Editor: Hartmut Vodermaier
Transaction Report:
(Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity,
letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this
compilation.)
1st Editorial Decision
03 October 2012
Thank you for submitting your manuscript on cohesin entry and exit gates in human cells for our
consideration. After some delay associated with the evaluation of back-to-back submissions, we
have now received the feedback of two expert referees. As you will see from these comments, these
referees would in principle consider the demonstration of conservation of both yeast cohesin gates in
mammalian cells of major importance. Nevertheless, they both remain unconvinced that the
presented dataset here is sufficiently decisive to strongly support such conclusions. In particular, the
reviewers criticize insufficient data/controls and incomplete documentation of the utilized
experimental settings on various instances. While the study is therefore currently not a good
candidate for publication in our journal, I realize that you may well be in a position to satisfactorily
address these predominantly technical concerns, and would for this reason be willing to allow you
an opportunity to respond to the reviewers in the form of a revised version of this manuscript. I have
to however stress that it is our policy to allow a single round of major revision only, and that it will
be essential to fully answer to the referees' points at this stage for eventual acceptance of the paper.
Referee 2 (point 4) also makes some further-reaching experimental suggestions that would clearly
make this study a more compelling overall advance, but given the requirements for major revision
efforts for the currently presented data, I understand that such investigations (unless already ongoing
in your lab) may be beyond the scope of the present study. From an editorial point of view, I feel it
would be important to mention the recent work on the yeast cohesin exit gate upfront in the
introduction section, rather than only brushing on it in the discussion; furthermore, please try to also
improve the overall writing of the study, and make sure to include brief Author Contribution and
Conflict of Interest statements.
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We generally allow for three months of major revision, and it is our policy that related or competing
manuscripts published during this standard revision period will have no negative impact on our final
assessment of your revised study. Nevertheless, we would in this case hope that you could resubmit
a consolidated study as early as possible, and I would therefore appreciate if you could get back to
me with some feedback on the feasibility of addressing the referees' points and with an outlook on
possible resubmission timelines here. Should you have any other questions regarding this decision
or the referee reports, please also do not hesitate to contact me.
Thank you again for the opportunity to consider this work. I look forward to receiving your revised
manuscript.
-----------------------------------------------Referee #1 (Remarks to the Author):
In this study, Buheitel & Stemmann investigate the requirements for cohesin loading in G1 and
cohesin unloading in prophase in human cells in terms of interactions between the subunits of the
tripartite cohesin ring (Smc1-Smc3-Scc1). They take advantage of a system that promotes the
formation of a very stable interaction between two proteins in a rapamycin-dependent manner
through tagging the corresponding proteins with FRB and FKBP. By knocking down endogenous
cohesin subunits while expressing the tagged versions they can analyze the effect of not allowing
disengagement of each one of the three so-called gates of the complex on its loading and unloading.
A requirement for disengagement of Smc1-Smc3 hinges for cohesin loading has been previously
shown in yeast (Gruber 2006) whereas the requirement for disengagement of Smc3-Scc1 interface
for cohesin release has been very recently demonstrated for the turnover of cohesin at pericentic
chromatin during mitosis in S. cerevisiae (Chan 2012). Nevertheless, the results of the current study
would provide proof of the conservation of the loading mechanism in human cells and would also
support the hypothesis that the mechanism releasing cohesin in prophase in metazoa is essentially
similar to the mechanism that regulates turnover of cohesin on chromatin at other times of the cell
cycle. Thus, the results presented in this study are potentially interesting. Unfortunately, I find many
important controls and additional data missing so that in its present form I cannot support its
publication.
Instead of showing the phenotype of Wapl depletion (figure 1), already well established, the authors
need to thoroughly characterize the context in which their experiments are performed. They should
present western blots showing expression of the tagged proteins, downregulation of the endogenous
proteins by siRNA, presence of these complexes in interphase chromatin and whether it is affected
by addition of rapamycin. It is of particular importance to show that formation of a link between
Smc3 and Scc1 does not interfere at least with Pds5 and Wapl recruitment. Additional regulators of
the prophase pathway like localization of Sgo1 should also be checked as control.
Figure by Figure
-Figure 2D. Show the expression levels of the tagged proteins with respect to the endogenous.
I would like to see immunoprecipitation experiments showing that the tagged proteins can
incorporate into a bonafide cohesin complex that can interact with SA, Pds5 and Wapl both in the
presence or absence of rapamycin.
Alternatively, the authors could show that cohesion defects found upon depleting the corresponding
endogenous proteins can be rescued by expression of the tagged proteins.
- Figure 3. Show western blots for the 3 different experimental set ups in which two endogenous
proteins are knocked down and the corresponding tagged proteins are expressed. Also, before
showing that the proteins are released in mitosis, it is important to show that they have been loaded
in the preceding interphase. In this regard, what happens to the Smc3intFRB+Smc1-intFKBP? I
assume that cohesin complexes can be loaded before rapamycin is added but over the next 30 h
before analysis loading is prevented and given the dynamic behaviour of cohesin in interphase it is
most likely that little cohesin is actually left on chromatin by the time prophase release starts.
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- Figure 4.
A. The quality of these data should be improved. This should be shown as part of Figure 2.
B-D. Now the system changes to a fusion of Smc1-Scc1. Again, the authors should show all the
required controls mentioned above, including ability to interact with the other cohesin subunits. Are
endogenous proteins being knocked out in Figure 4D? Show the western blots. Show loading of
cohesin in interphase in the absence of endogenous complexes before concluding that release is fine.
- Figure 5.
Show the western blots for the knock downs and expression of the tagged proteins. Show staining
for cohesin subunits other than tagged Smc1 or Smc3.
Other minor points:
-Figure 1 is totally unnecessary since it is just a duplication of previously published data and should
be deleted to leave room for the data requested.
- Why are there 2 bands in lane 2 of western blot with FKBP in figure 2D?
Regarding references:
- In page 3 of Introduction, "the kollerin complex, which in vertebrates is recruited to DNA in
telophase by prereplicative complexes". This is not correct. The requirement of preRC for cohesin
loading has been demonstrated in Xenopus by the Hirano and Walter labs, but it does not occur in
human cells (Guillou 2010).
-"Prophase pathway signalling involves the releasin complex, kinase activity of Plk and
phosphorylation of SA2". Shintomi 2009 should be cited here, since it is the only study showing that
Pds5 is required for prophase release. Alternatively, substitute "releasin complex" by "Wapl". Also,
both Plk1 and Aurora B kinases are required for this pathway in Xenopus (Losada 2002) and in
human cells (GimÈnez-Abian 2004). Finally, please note that phosphorylation affects both SA1 and
SA2 (Losada 2002; Sumara 2002).
Referee #2 (Remarks to the Author):
Faithful segregation of chromosomes during mitosis requires sister chromatid cohesion mediated by
cohesin complex through entrapping sister DNAs inside the tripartite ring. Experiments in yeast
have suggested that cohesin undergoes DNA entrapment and release dynamically, through transient
opening of Smc1/3 interface and Smc3/alpha-kleisin (Scc1) interface, respectively. It is well known
that DNA is released from cohesin by the proteolytic cleavage of kleisin upon anaphase onset, but
the proteolysis-independent dissociation of cohesin in prophase/prometaphase through what it is
called prophase pathway is not well understood.
This paper addresses in mammalian cells if cohesin release during the prophase pathway is through
the Smc3/Scc1 gate and if cohesin's DNA entrapment is through the Smc1/3 gate. The authors
utilized Rapamycin-inducible heterodimerization of FRB and FKBP, in order to conditionally tether
the pairwise interactions between Smc1, Smc3 and Scc1 in synchronized cell populations. They
essentially described that cohesin persisted on prometaphase chromosomes by tethering the
Smc3/Scc1 interface, and loading of cohein onto chromosomes in telophase was blocked by
tethering the Smc1/3 interface. Based on these results the authors concluded that the joining and
disjoining of sister chromatids involves DNA to enter and exit the cohesin ring through different
gates. This is an important step towards understanding of cohesin regulation, as it implies that the
mechanism promoting DNA entrapment and release is universally conserved from yeast to human.
However, I have some concerns, which needs to be clarified to be supportive for the publication, as
detailed below. Particularly, as conclusions are consistent with what has been revealed in yeast
works, I consider it important to carefully control the experimental settings.
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Major comments:
1. To identify the gate of cohesin ring for DNA entry and exit in mammalian cells, the authors
engineered the cell lines expressing FRB/FKBP-tagged series of cohesin subunits, whose tethering
can be induced by rapamycin treatment. Here it is essential to show rapamycin-mediated fused
proteins by immunoblotting, not only for Scc1-Smc1 (Figure 4B) but also for Scc1-Smc3 and
Smc1/3. These latter two combinations are apparently more important to show, as they block normal
cohesin dynamics. I expect to see them in one gel encompassing both Smc1/3 or Scc1 and their
fused versions, rather than cropped bands, because it allows us to estimate the efficiency of the
tethering by rapamycin. I am also curious to see if Scc1-Smc3 and Smc1/3 chimeras also co-purifies
with chromatin like Scc1-Smc does (as shown in Figure 4C).
2. The authors show a Wapl-like phenotype only when FKBP-Scc1 and Smc3-FRB were expressed
together and rapamycin was present. I prefer to exclude the possibility if the preservation of
cohesion on chromosome arms is not due to any perturbation of the interaction of cohesin with
Wapl, due to any potential conformational change in the presence of the FKBP-Scc1 and Smc3-FRB
tagged proteins. The authors should be able to preclude these formal possibilities by
immunoprecipitation assays for cohesin, Pds5 and Wapl.
3. I find the naming of the chromosome morphology in spread sample confusing. What they called
"zipped" chromosomes seems to refer tightly cohesed chromosomes, as seen after depletion of
Wapl. What makes me confusing is that the chromosomes in cells expressing Scc1-Smc1 tethered
protein appears to also have a tight cohesion of sisters, but here they are not categorized as "zipped"
(Figure 5E). Whether they have less cohesed sisters is not very clear and it makes difficult to claim
that tethering Scc1-Smc1 do not affect the release of cohesin. Can the authors assess the
chromosome morphology after nocodazole-treatment, which will clarify the situation in tethering
Scc1-Smc1 whether sister chromatids are completely dissociated from each other or not.
4. Having nice tools to control two gates of the cohesin ring, it provides the authors unique
opportunity to ask if the loading (kollelin) and the releasing (releasin) activity might change during
mitosis. These questions are of high interest for the cohesin field, and would make the paper very
different from the related yeast works.
Minor comment:
In introducing Wapl, Gandhi 2006 should be cited along with Kueng 2006 (page 5).
1st Revision - authors' response
11 December 2012
Please find enclosed our revised manuscript with the title: "Prophase Pathway Dependent Removal
of Cohesin from Human Chromosomes Requires Opening of the Smc3-Scc1 Gate". First of all, we
would like to thank you and the referees for the thorough study of our manuscript and the
constructive criticism. Our efforts to further strengthen our conclusions have resulted in new figures
2E, 3A-C, 5C+D, 6C+D and S1 within the revised manuscript. The key improvements are the
following:
(1) The revised manuscript now contains Western blots that illustrate both the siRNA mediated
depletion of the endogenous proteins and, at the same time, the induced expression of the transgenes
(new figure 2E). We now demonstrate that our FRB/FKBP-tagged pairs of cohesin subunits replace
their endogenous counterparts as the main species in the cell. (Please note that the Western signals
for Scc1-FRB and Smc3-FRB do not accurately reflect their expression levels because the available
antibodies display a greatly reduced sensitivity when their antigens are C-terminally tagged.)
(2) The functionality of our engineered cohesin complexes is underscored. More specifically, we
employ immunofluorescence-microscopy on pre-extracted interphase cells and Western analyses of
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isolated interphase chromatin to now demonstrate that all FRB-tagged cohesin subunits retain the
ability to associate with DNA (new figures 3C and S1).
(3) We had reported that the prophase-specific dissociation of cohesin from chromosome arms
requires passage of DNA through the Smc3-Scc1 gate. This claim was based on the fact that cohesin
persisted on mitotic chromatin when Smc3-FRB and FKBP-Scc1 were linked by rapamycin. This
most important finding of our work has somewhat been challenged by the reviewers who suspected
that the tags might interfere with proper interaction of prophase pathway components with the
engineered cohesin. Here, it is important to stress that the corresponding cohesin was removed
normally in the absence of rapamycin demonstrating that the tags did not interfere with the prophase
pathway per se. Nevertheless, the tags might interfere with the action of prophase components only
upon their rapamycin-induced heterodimerisation. Additional experiments now rule out even this
remote possibility. Co-immunoprecipiations demonstrate that the FRB/FKBP-tagged cohesin
subunits (as well as the Scc1-Smc1 fusion) retain the ability to interact with each other, with
endogenous cohesin components, and with Wapl (new figures 3A, 3B, 5C and 5D). Importantly,
these interactions are unaffected by the presence or absence of rapamycin, which further
corroborates our key discovery.
We hope that with these improvements we have sufficiently strengthened our main points and that
our manuscript will now be suitable for publication in The EMBO Journal.
Below please find our detailed point-by-point response to all major and minor comments raised by
the expert referees.
Thank you again for your time and consideration.
Point-by-point response to the referees' comments
First of all, we would like to thank the referees for their thorough evaluation of our work and their
constructive criticism, which we have addressed as outlined in detail below:
Referee 1:
Major Points
"Figure 2D. Show the expression levels of the tagged proteins with respect to the endogenous.
I would like to see immunoprecipitation experiments showing that the tagged proteins can
incorporate into a bonafide cohesin complex that can interact with SA, Pds5 and Wapl both in the
presence or absence of rapamycin.
Alternatively, the authors could show that cohesion defects found upon depleting the corresponding
endogenous proteins can be rescued by expression of the tagged proteins."
The requested experiments address important issues and we are grateful to the reviewer for pointing
them out. The new figure 2E of the revised manuscript now documents that the siRNA mediated
depletions of the endogenous cohesin subunits work appreciably well and, furthermore, that the
endogenous proteins are largely replaced by FRB/FKBP-tagged variants when RNAi is combined
with transgene induction. It should be stressed that for the detection of Smc3 and Scc1 we were
limited to C-terminus specific antibodies. Given that both antibodies exhibit a greatly reduced
affinity for C-terminally tagged versions of their antigens, the weak Western signals for Smc3-FRB
and Scc1-FRB represent an underestimation of their actual expression levels.
To demonstrate the retained ability of our tagged cohesin subunits to be incorporated into a bona
fide cohesin complex, we performed various immunoprecipitation experiments in the absence and
presence of rapamycin and using anti-FRB- and anti-Wapl antibodies (see new figures 3A and -B,
respectively). Based on these experiments we can report that the tagged cohesin subunits not only
interact with each other, but also with the endogenous third ring component (e.g. endogenous Smc1
in the case of FKBP-Scc1 and Smc3-FRB), SA1, and Wapl.
Referee #1 especially emphasised the importance of showing that formation of a link between Smc3
and Scc1 does not interfere with recruitment of the essential prophase pathway component Wapl.
The left panel of the new figure 3B now shows exactly this, i.e. co-IP of FKBP-Scc1 and Smc3-FRB
with Wapl irrespective of whether or not heterodimerisation has been induced by rapamycin.
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(We would like to point out that in the Smc3-FRB and FKBP-Scc1 expressing cell line the
previously detectable FRB signal (see below) disappears from chromatin at the beginning of mitosis
when rapamycin is not present. This strongly argues that the engineered cohesin complex remains
susceptible to the prophase pathway despite its tagging. We consider it unlikely that the FRB/FKBPtags interfere with the action of prophase components only upon their rapamycin-induced
heterodimerisation.)
"Figure 3. Show western blots for the 3 different experimental set ups in which two endogenous
proteins are knocked down and the corresponding tagged proteins are expressed. Also, before
showing that the proteins are released in mitosis, it is important to show that they have been loaded
in the preceding interphase. In this regard, what happens to the Smc3intFRB+Smc1-intFKBP? I
assume that cohesin complexes can be loaded before rapamycin is added but over the next 30 h
before analysis loading is prevented and given the dynamic behaviour of cohesin in interphase it is
most likely that little cohesin is actually left on chromatin by the time prophase release starts."
We thank the reviewer for suggesting these important controls as well as for pointing out the caveat
regarding the experiments with our "hinge"-cell line. We have now documented the knockdown of
the endogenous as well as the expression of the exogenous cohesin subunits (new figure 2E). This
additional experiment was performed by a regime virtually identical to the one used for the
experiments shown in figure 4 of the revised manuscript: The same amount of cells were transfected
with the same siRNAs using identical concentrations and knockdown/expression was carried out for
three days as before. We would like to add that the knockdown efficiency should not have a strong
impact on our findings, at least qualitatively. This is because 1) there is almost no background
staining in our experiments due to wild type cohesin being virtually undetectable on prometaphase
chromatin (figure 4C) and 2) we are looking directly at the behavior of our transgene encoded
cohesin via a FRB specific antibody (figure 4D).
We agree with the reviewer that it is important to first prove the proper loading of our engineered
cohesin complexes onto chromatin before considering their dissociation in mitotic prophase. The
new figure S1 now shows that the FRB-tagged cohesin subunit gets loaded onto interphase
chromatin in each of the three double-transgenic cell lines. To further corroborate this notion we
knocked down endogenous cohesin and simultaneously induced expression of the respective
transgenes for three days (much like the experiments shown in figure 4). We then prepared
chromatin-enriched as well as cytoplasmic fractions and performed Western blots to detect
FRB/FKBP-tagged as well as endogenous cohesin subunits. The corresponding results are shown in
the new figure 3C and confirm that the tagged cohesin subunits behave just like the endogenous
counterparts with respect to their pronounced association with interphase chromatin.
Reviewer #1 rightfully points out that - based on its dynamic behavior - cohesin, which is prevented
from opening its Smc1-Smc3 hinge, might quantitatively be lost from interphase chromatin simply
because re-loading is blocked. Then, the inability to detect FRB positive prometaphase chromatin in
Smc1-int.FKBP and Smc3-int.FRB expressing cells in the presence of rapamycin could not be used
as an argument to rule out the hinge as the DNA exit gate. However, two observations of ours argue
against this scenario: 1) If DNA would exit through the Smc1-Smc3 gate, then one would expect
persistence of some Smc1-int.FKBP and Smc3-int.FRB containing cohesin on prometaphase
chromatin as well as an increased number of zipped chromosomes in the experiments, in which
rapamycin was added only late, i.e. only about four hours before mitotic entry and seven hours prior
to fixation. As figures 4B, -E and -F show, this is not the case. 2) In the experiments, in which
Hek293 cultures were treated with rapamycin and thymidine for 16 hours and then released into
rapamycin containing medium for 14 hours, we readily detect FRB positive interphase chromatin in
Smc1-int.FKBP and Smc3-int.FRB expressing cells that have not yet progressed into mitosis (new
figure S1).
"Figure 4.
A. The quality of these data should be improved. This should be shown as part of Figure 2."
We now show improved immunoprecipitation experiments, which demonstrate the rapamycinindependent interaction of the transgene encoded FRB/FKBP-tagged proteins with each other, with
endogenous cohesin subunits, and with Wapl (see new figures 3A and -B).
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"B-D. Now the system changes to a fusion of Smc1-Scc1. Again, the authors should show all the
required controls mentioned above, including ability to interact with the other cohesin subunits. Are
endogenous proteins being knocked out in Figure 4D? Show the western blots. Show loading of
cohesin in interphase in the absence of endogenous complexes before concluding that release is
fine."
Is the Scc1-Smc1 fusion protein functional? In the revised version we included immunoprecipitation
experiments that show incorporation of the Scc1-Smc1 fusion in a complex with endogenous Smc3.
Since the Scc1-Smc1 fusion was not tagged, we used an anti-Smc3 antibody to immunoprecipitate
endogenous Smc3 in cells, in which endogenous Scc1 and Smc1 were depleted by RNAi. The
results show that Smc3 interacts with the Scc1-Smc1 fusion protein, SA1 and Wapl (new figure 5C).
Since, we could not fully exclude that the association with Wapl is bridged via remaining
endogenous Scc1, we validated this result by an anti-Wapl immunoprecipitation, which revealed coprecipitation of Scc1-Smc1 fusion (new figure 5D). Thus, the Scc1-Smc1 fusion protein is
incorporated into cohesin complexes. But does it also get loaded onto DNA? Due to lack of a fusion
protein specific tag, we cannot specifically assess loading of the Scc1-Smc1 fusion protein onto
chromatin via IF. Therefore, we demonstrated its ability to bind to DNA via chromatin isolation
from unsynchronised, i.e. largely interphasic, cells (figure 5B). Based on our analyses, we conclude
that the human Scc1-Smc1 fusion protein retains functionality, which is consistent with the fact that
a corresponding fusion can substitute for Scc1 and Smc1 in S. cerevisiae (Gruber et al., 2006).
Were endogenous Scc1 and Smc3 depleted? We apologise for the lack of clarity regarding the
experimental settings in the original manuscript. The corresponding endogenous proteins had, in
fact, been depleted by RNAi in the experiment for the original figure 4D (now 5E). The general
knock-down efficiency for Scc1 and Smc1 can be appreciated by looking at figure 2E. Figure 5A
demonstrates the expression level of the Scc1-Smc1 fusion with respect to remaining endogenous
Smc1 after two days of siRNA mediated depletion. This illustrates that the expression level of the
fusion protein is low (although suboptimal blotting efficiency of this large construct, which migrates
with an apparent molecular weight of 250 kDa in SDS-PAGE, might result in an underestimation of
its real amount). However, a very small fraction of the cell's total cohesin is sufficient to maintain
largely normal cohesion. Moreover, the background in our experiments is negligible (meaning that
practically all mitotic cells are devoid of any cohesin staining). It is for these reasons that we would
expect to see at least a small effect of Scc1-Smc1 expression if disengagement of this gate was
indeed required for DNA to leave the ring.
"- Figure 5.
Show the western blots for the knock downs and expression of the tagged proteins. Show staining for
cohesin subunits other than tagged Smc1 or Smc3."
We document the knockdown efficiency of our siRNAs in the new figure 2E. As mentioned above,
we do stain for the internal FRB tag of our transgene encoded Smc3, which enables us to selectively
assess chromatin association of the engineered cohesin without any background from the
endogenous protein.
As suggested by the reviewer, we reiterated the experiment and performed a co-staining of
exogenous Smc3-int.FRB and endogenous Scc1. This experiment confirmed that the amount of FRB
positive G1 chromatin drops (by about 30%) when cells, in which Smc1-int.FKBP and Smc3int.FRB largely replaced endogenous Smc1 and -3, exit mitosis in the presence of rapamycin. At the
same time, the rate of reloading of endogenous Scc1 remained unchanged at around 80% (new
figure 6C and -D). This means that rapamycin had an impact only on the engineered cohesin and not
on remaining endogenous complexes. (While the Scc1 signals remained unaffected in respect to the
number of cells that exhibited reloading, the intensity of its fluorescence signal per individual cell
did, in fact, drop by 15% in presence of rapamycin (data not shown). This is consistent with the
transgenic proteins interacting with endogenous Scc1.)
Minor Points
"Figure 1 is totally unnecessary since it is just a duplication of previously published data and should
be deleted to leave room for the data requested."
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Again, we agree with the reviewer. However, as this figure is suitable to introduce the layman reader
into the general mechanism of the prophase pathway and as it offers comparison of phenotypes with
the subsequent figures, we would like to keep it as part of the manuscript (although it could easily be
moved into the supplement).
"Why are there 2 bands in lane 2 of western blot with FKBP in figure 2D?"
We are not sure about the nature of the faster migrating band. All we can say is that based on
restriction analysis, DNA-sequencing and transient transfection experiments the corresponding
transgene expression construct is error-free. It should also be mentioned that this double band is not
a unique feature of one particular clone but can be seen in all clones out of several independent
isolates.
"In page 3 of Introduction, "the kollerin complex, which in vertebrates is recruited to DNA in
telophase by prereplicative complexes". This is not correct. The requirement of preRC for cohesin
loading has been demonstrated in Xenopus by the Hirano and Walter labs, but it does not occur in
human cells (Guillou 2010)."
We thank the referee for pointing this out and apologise for our negligence. We have corrected this
passage in the revised manuscript.
"Prophase pathway signalling involves the releasin complex, kinase activity of Plk and
phosphorylation of SA2". Shintomi 2009 should be cited here, since it is the only study showing that
Pds5 is required for prophase release. Alternatively, substitute "releasin complex" by "Wapl". Also,
both Plk1 and Aurora B kinases are required for this pathway in Xenopus (Losada 2002) and in
human cells (Giménez-Abian 2004). Finally, please note that phosphorylation affects both SA1 and
SA2 (Losada 2002; Sumara 2002)."
We apologise for the imprecise statements and have now rectified the corresponding text segments.
Referee 2:
Major comments
"To identify the gate of cohesin ring for DNA entry and exit in mammalian cells, the authors
engineered the cell lines expressing FRB/FKBP-tagged series of cohesin subunits, whose tethering
can be induced by rapamycin treatment. Here it is essential to show rapamycin-mediated fused
proteins by immunoblotting, not only for Scc1-Smc1 (Figure 4B) but also for Scc1-Smc3 and
Smc1/3. These latter two combinations are apparently more important to show, as they block
normal cohesin dynamics. I expect to see them in one gel encompassing both Smc1/3 or Scc1 and
their fused versions, rather than cropped bands, because it allows us to estimate the efficiency of the
tethering by rapamycin. I am also curious to see if Scc1-Smc3 and Smc1/3 chimeras also co-purifies
with chromatin like Scc1-Smc does (as shown in Figure 4C)."
We apologise for the lack of clarity regarding our description of the nature of the rapamycinmediated connection between transgene encoded pairs of cohesin subunits. While for most
experiments we use the FRB/FKBP-system for inducible dimerisation, the fusion of the two cohesin
subunits in former Figure 4B (now 5A) was created by actually fusing the open reading frames of
Scc1 and Smc1 to create a construct which is expressed as one protein. This in-frame fusion features
a covalent bond between the two subunits, which is SDS-resistant. Unfortunately the rapamycinmediated FRB-FKBP dimerisation cannot withstand SDS treatment and therefore it is not possible
to visualise the other dimers on an SDS gel.
Nonetheless, we fully agree with the reviewer that we have to prove that the FRB/FKBP-tagged
cohesin variants retain the ability to bind to chromatin in interphase and that this association is
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independent of rapamycin. We are happy to present a newly conducted experiment, in which we
prepared a chromatin-enriched as well as a cytoplasmic fraction. Western analyses of these samples
demonstrate that all transgenic cohesin subunits are, indeed, able to bind to chromatin and do so
favorably (new figure 3C).
"The authors show a Wapl-like phenotype only when FKBP-Scc1 and Smc3-FRB were expressed
together and rapamycin was present. I prefer to exclude the possibility if the preservation of
cohesion on chromosome arms is not due to any perturbation of the interaction of cohesin with
Wapl, due to any potential conformational change in the presence of the FKBP-Scc1 and Smc3-FRB
tagged proteins. The authors should be able to preclude these formal possibilities by
immunoprecipitation assays for cohesin, Pds5 and Wapl."
This is an excellent remark and we immediately agreed that our studies would strongly profit from
these additional experiments. Therefore, we performed immunoprecipitation assays using anti-FRB
and anti-Wapl antibodies to unequivocally demonstrate that our transgenes can be incorporated into
a complex with other cohesin subunits and Wapl (new figures 3A and -B).
"I find the naming of the chromosome morphology in spread sample confusing. What they called
"zipped" chromosomes seems to refer tightly cohesed chromosomes, as seen after depletion of Wapl.
What makes me confusing is that the chromosomes in cells expressing Scc1-Smc1 tethered protein
appears to also have a tight cohesion of sisters, but here they are not categorised as "zipped"
(Figure 5E). Whether they have less cohesed sisters is not very clear and it makes difficult to claim
that tethering Scc1-Smc1 do not affect the release of cohesin. Can the authors assess the
chromosome morphology after nocodazole-treatment, which will clarify the situation in tethering
Scc1-Smc1 whether sister chromatids are completely dissociated from each other or not."
Again, we apologise for not making our experimental procedure fully plain. In the experiment
shown in former figure 4E (figure 5E of the revised manuscript) the cells were actually arrested in
nocodazole but due to technical reasons were kept in this arrest for a rather short time (≤ 3 h). After
such a short prometaphase arrest the chromosome morphology of cohesed "butterfly-like"
chromosomes is often not as pronounced as it is after prolonged incubation in nocodazole. However,
chromosomes with separated arms are still easily discernable under the microscope from those,
which remain tightly cohesed along their entire length. We agree that the example shown in that
figure was unfortunate, so we revisited the experiment and replaced the corresponding image with a
more appropriate one in the revised manuscript.
"Having nice tools to control two gates of the cohesin ring, it provides the authors unique
opportunity to ask if the loading (kollelin) and the releasing (releasin) activity might change during
mitosis. These questions are of high interest for the cohesin field, and would make the paper very
different from the related yeast works."
We would like to thank the referee for suggesting this interesting set of experiments. Although we
agree that assessing the loading and releasing activity during mitosis would be of great value for the
field, we fear that the accompanying experiments are beyond the scope of the study presented here.
Nonetheless, we are eager to address these issues during our future studies.
Minor comments
"In introducing Wapl, Gandhi 2006 should be cited along with Kueng 2006 (page 5)."
The citation was added to the revised manuscript and can be found on pages 4 and 6.
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Acceptance letter
08 January 2013
Thank you for submitting your revised manuscript for our consideration. Please excuse the delay in
getting back to you with a response in this case: While one of the original reviewers appeared fully
satisfied with your revision, the other one retained serious concerns and reservations about potential
acceptance of this work (see comments copied below). I therefore decided to myself closely look
into your responses, and to additionally involve an expert editorial advisor as arbitrating referee. We
have now heard back from this third expert, and I am pleased to communicate that this expert
considered your responses to the initial round of review on the whole satisfactory. Furthermore, the
arbitrator felt that while some of the remaining concerns of referee 1 are well-taken, they would not
provide overriding reasons to further delay publication of the manuscript in light of the combined
body of evidence presented to support the conclusions of the study.
I am therefore happy to accept the manuscript for publication in The EMBO Journal at this point!
Thank you again for your contribution to The EMBO Journal and congratulations on a successful
publication. Please consider us again in the future for your most exciting work.
----------------------Referee reports on revised manuscript:
Referee #1
(Remarks to the Author)
Although the authors have addressed some of my concerns in the revised manuscript, I still do not
find this version strong enough for publication in EMBO J. I believe that further experiments have
to be done and the quality of some data has to be improved before publication.
"Figure 2D. Show the expression levels of the tagged proteins with respect to the endogenous.
I would like to see immunoprecipitation experiments showing that the tagged proteins can
incorporate into a bonafide cohesin complex that can interact with SA, Pds5 and Wapl both in the
presence or absence of rapamycin.
Alternatively, the authors could show that cohesion defects found upon depleting the corresponding
endogenous proteins can be rescued by expression of the tagged proteins."
A. The requested experiments address important issues and we are grateful to the reviewer for
pointing them out. The new figure 2E of the revised manuscript now documents that the siRNA
mediated depletions of the endogenous cohesin subunits work appreciably well and, furthermore,
that the endogenous proteins are largely replaced by FRB/FKBP-tagged variants when RNAi is
combined with transgene induction. It should be stressed that for the detection of Smc3 and Scc1 we
were limited to C-terminus specific antibodies. Given that both antibodies exhibit a greatly reduced
affinity for C-terminally tagged versions of their antigens, the weak Western signals for Smc3-FRB
and Scc1-FRB represent an underestimation of their actual expression levels.
R. The expression levels of most proteins is relay low. If the authors suspect that the presence of the
tag interfere with detection with antibodies raised against C-terminal antigens, they should use
different antibodies. How can they estimate abundance otherwise? For Smc3, even when the tag is
near the hinge the amount of expressed protein is low compared with endogenous levels. Also, when
performing a depletion+add back it is important to load a "standard" with different amounts of
extract to be able to estimate in a more quantitative way how much protein the cell has left.
A. To demonstrate the retained ability of our tagged cohesin subunits to be incorporated into a bona
fide cohesin complex, we performed various immunoprecipitation experiments in the absence and
presence of rapamycin and using anti-FRB- and anti-Wapl antibodies (see new figures 3A and -B,
respectively). Based on these experiments we can report that the tagged cohesin subunits not only
interact with each other, but also with the endogenous third ring component (e.g. endogenous Smc1
in the case of FKBP-Scc1 and Smc3-FRB), SA1, and Wapl.
R. I do not think this experiment is done properly. The authors should have included western blots
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with Wapl and Pds5 in the analysis of the immunoprecipitates. Why the levels of SA in the input are
reduced in +rapa with respect to -rapa?
A. Referee #1 especially emphasised the importance of showing that formation of a link between
Smc3 and Scc1 does not interfere with recruitment of the essential prophase pathway component
Wapl. The left panel of the new figure 3B now shows exactly this, i.e. co-IP of FKBP-Scc1 and
Smc3-FRB with Wapl irrespective of whether or not heterodimerisation has been induced by
rapamycin.
(We would like to point out that in the Smc3-FRB and FKBP-Scc1 expressing cell line the
previously detectable FRB signal (see below) disappears from chromatin at the beginning of mitosis
when rapamycin is not present. This strongly argues that the engineered cohesin complex remains
susceptible to the prophase pathway despite its tagging. We consider it unlikely that the FRB/FKBPtags interfere with the action of prophase components only upon their rapamycin-induced
heterodimerisation.)
R. Regarding the immunoprecipitation with Wapl in Figure 3B, I'm afraid that the quality of these
results is rather low. The choice of Wapl is maybe not so appropriate since it appears not to be a
"stoichiometric" component of the complex (at least in yeast). Probably Pds5 or SA would have
been a better choice. Although the authors find some tagged proteins in the Wapl
immunoprecipitates, the only way to know whether this amount is "normal" and thereby sufficient to
perform properly the prophase pathway is to show a quantitative comparison of endogenous
complex and tagged complexes. Also, I do not understand why they choose to do western blot
analysis with the less abundant of the cohesin complex components, SA1 (SA2 is 10 times more
abundant in human cells!).
R. "Figure 3. Show western blots for the 3 different experimental set ups in which two endogenous
proteins are knocked down and the corresponding tagged proteins are expressed. Also, before
showing that the proteins are released in mitosis, it is important to show that they have been loaded
in the preceding interphase. In this regard, what happens to the Smc3intFRB+Smc1-intFKBP? I
assume that cohesin complexes can be loaded before rapamycin is added but over the next 30 h
before analysis loading is prevented and given the dynamic behaviour of cohesin in interphase it is
most likely that little cohesin is actually left on chromatin by the time prophase release starts."
A. We agree with the reviewer that it is important to first prove the proper loading of our engineered
cohesin complexes onto chromatin before considering their dissociation in mitotic prophase. The
new figure S1 now shows that the FRB-tagged cohesin subunit gets loaded onto interphase
chromatin in each of the three double-transgenic cell lines. To further corroborate this notion we
knocked down endogenous cohesin and simultaneously induced expression of the respective
transgenes for three days (much like the experiments shown in figure 4). We then prepared
chromatin-enriched as well as cytoplasmic fractions and performed Western blots to detect
FRB/FKBP-tagged as well as endogenous cohesin subunits. The corresponding results are shown in
the new figure 3C and confirm that the tagged cohesin subunits behave just like the endogenous
counterparts with respect to their pronounced association with interphase chromatin.
R. The new results presented in the revised manuscript (Figure 3C and Figure S1) show that the
tagged complexes do load on interphase chromatin. That is fine. I also understand the author's
argument about Smc1-Smc3 hinge not being an exit gate. However, I still do not understand why
Smc3intFRB+Smc1-intFKBP can be detected on chromatin. Maybe this is not the only entry gate?
R. "B-D. Now the system changes to a fusion of Smc1-Scc1. Again, the authors should show all the
required controls mentioned above, including ability to interact with the other cohesin subunits. Are
endogenous proteins being knocked out in Figure 4D? Show the western blots. Show loading of
cohesin in interphase in the absence of endogenous complexes before concluding that release is
fine."
A. Is the Scc1-Smc1 fusion protein functional? (...) Were endogenous Scc1 and Smc3 depleted? We
apologise for the lack of clarity regarding the experimental settings in the original manuscript. The
corresponding endogenous proteins had, in fact, been depleted by RNAi in the experiment for the
original figure 4D (now 5E). The general knock-down efficiency for Scc1 and Smc1 can be
appreciated by looking at figure 2E. Figure 5A demonstrates the expression level of the Scc1-Smc1
fusion with respect to remaining endogenous Smc1 after two days of siRNA mediated depletion.
This illustrates that the expression level of the fusion protein is low (although suboptimal blotting
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efficiency of this large construct, which migrates with an apparent molecular weight of 250 kDa in
SDS-PAGE, might result in an underestimation of its real amount). However, a very small fraction
of the cell's total cohesin is sufficient to maintain largely normal cohesion. Moreover, the
background in our experiments is negligible (meaning that practically all mitotic cells are devoid of
any cohesin staining). It is for these reasons that we would expect to see at least a small effect of
Scc1-Smc1 expression if disengagement of this gate was indeed required for DNA to leave the ring.
R. I think that the authors cannot make any conclusion from this experimental set up. The levels of
Scc1-Smc1 fusion protein are much lower than what is left of endogenous Smc1 after 2 days of
siRNA. They say that the efficiency of the siRNA is similar to that of Figure 2E (an unrelated
experiment using the same siRNA). This means that the actual amount of complexes containing
Smc1-Scc1 fused is so low that even if this fusion would affect the exit gate this would not be seen
by immunofluorescence. The authors should make a different construct tagging the fusion protein so
that they can see how it loads in interphase and unloads in mitosis.
R. "- Figure 5.
Show the western blots for the knock downs and expression of the tagged proteins. Show staining
for cohesin subunits other than tagged Smc1 or Smc3."
A. As suggested by the reviewer, we reiterated the experiment and performed a co-staining of
exogenous Smc3-int.FRB and endogenous Scc1. This experiment confirmed that the amount of FRB
positive G1 chromatin drops (by about 30%) when cells, in which Smc1-int.FKBP and Smc3int.FRB largely replaced endogenous Smc1 and -3, exit mitosis in the presence of rapamycin. At the
same time, the rate of reloading of endogenous Scc1 remained unchanged at around 80% (new
figure 6C and -D). This means that rapamycin had an impact only on the engineered cohesin and not
on remaining endogenous complexes. (While the Scc1 signals remained unaffected in respect to the
number of cells that exhibited reloading, the intensity of its fluorescence signal per individual cell
did, in fact, drop by 15% in presence of rapamycin (data not shown). This is consistent with the
transgenic proteins interacting with endogenous Scc1.)
R. The authors should show levels of endogenous and exogenous proteins for this particular
experiment, not refer again to the results in Fig.2E, which correspond to an unrelated regime.
Although I agree with the authors that the results shown in this figure indicate that "closing" the
Smc1-Smc3 hinge upon addition of rapamycin affects cohesin loading, the effect is only a two-fold
reduction in the number of cells. Together with my previous comments, it is not clear to me that this
is indeed an entry gate and/or at least not the only one. According to Figure S1 and 3C, there is
cohesin on interphase chromatin after closing any of the three potential gates. What happens to
loading in interphase (upon nocodazole arrest and release) when the other two gates are closed?
What happens when two out of three gates are closed?
Referee #2
(Remarks to the Author)
Comment to "Prophase pathway dependent removal of cohesin from human chromosomes" by
Buheitel and Stemmann.
In this revised manuscript, the authors have made great efforts to experimentally address the points I
raised on the original version. Most of them were requests to show the controls, that are now
adequately provided, in order to make the conclusion more convincing. I also appreciate it that the
texts are reorganized in a way that is more comprehensive for wider readership. Especially, I was
confused from the former version that Smc1-Scc1 is expressed as a fusion protein whereas
rapamycin-inducible tethering was utilized for the other combinations; but this was now clear. I find
that new Fig 5E is much suitable for the presentation to prevent any misunderstanding, like I did in
the previous version. Therefore, my original concerns are successfully addressed and do not have
any further requests before considering it for the publication.
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