Peer review of: Identifying proteins bound to native mitotic ESC chromosomes reveals chromatin repressors are i (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

In this manuscript, Djeghloul et al provide a proteomic characterization of mitotic chromatin using 
an elegant FACS approach to purify native mitotic chromosomes. They identify both new and 
known proteins associated with mitotic chromosomes in mouse ES cells and correlate the presence 
of typical repressors associated with heterochromatin to the compaction of the chromosomes. 
Since ES cells are known to display reduced heterochromatin, they further study the difference in 
compaction with various somatic cells, and show ES cells present particularly uncompact mitotic 
chromosomes. Overall, the technique is a good addition to the current state-of-the-art and the 
data interesting although mainly correlative. Below, I provide specific comments and criticisms 
that I think should be addressed before publishing a paper that may have the potential to change 
the way the field of mitotic bookmarking is approached. 

1/ The key advantage of the technique is to use unfixed, native chromosomes to describe the 
proteins that are bound. Indeed, several papers have now shown that fixatives may induce drastic 
miss-localization of proteins, particularly TFs, during mitosis. However, the authors have not 
clearly ruled out the potential issues of their technique: how can they ensure a massive loss of 
proteins is not occurring during this preparation and the sorting? I think it is very important the 
authors show in their purified chromosomes that the chromatin is maintained in the right 
configuration. I know they test some structural properties, which is important, but since they give 
a mitotic bookmarking focus to the paper it should be mandatory they provide the following 
genome-wide descriptions: 
• ATAC-seq to confirm that there procedure is not accompanied by a loss of chromatin accessibility 
at regulatory elements. The mitotic maintenance of significant accessibility has now been shown 
by several labs. 
• ChIP-seq of proven mitotic bookmarking factors in ES cells, such as Esrrb (Festuccia, 2016) and 
Tbp (Teves, 2018) 
If in their purified chromosomes these features are not fully preserved, then it will be important 
the authors describe and analyze the limitations of the technique. 

2/ The technique is based on comparing mitotic lysates and purified chromosomes. However, this 
may lead to major confusions for proteins that are partially or largely degraded during mitosis. 
Hence, the authors should analyze bulk asynchronous cells as well to avoid having false positives 
based on mitotic ratios exclusively. 

3/ Connected to point 1, the authors show a number of staining for several TFs previously shown 
to bind mitotic chromatin, such as Sox2. These stainings are not convincing at all, they barely 
show any signal on the chromosomes, while live imaging showed major enrichment on the 
chromosomes (Teves, 2016; De Luz, 2016). 

4/ My last point regarding the technique itself: the authors should provide a more principled 
comparison with the paper from Schubeler lab published in Nature Communications (Ginno, 2018): 
which proteins are differentially identified in both studies? For those proteins, what a live imaging 
approach would show? 

5/ In the experiments using DNMT TKO ES cells, the authors observe, as expected, that 
chromocenters lose K9me3 and gain K27me3 (although this is not described in the text). In 
contrast, this peri-centric enrichment for K27me3 is not seen in mitotic chromosomes. The authors 
should comment on this. 

6/ The authors conclude heterochromatic regulators participate to the compaction of mitotic 
chromosomes, based on the study of knock-outs. However, they do not control for a difference 
that may already be present in interphase (as they do later in the manuscript when they compare 

ES and somatic cells). It is important to understand, I believe, whether the defects of compaction 
are specific to mitotic chromosomes or to the chromatin in general. 

7/ The data on MeCP2 and K9me3 is really puzzling. The authors should show that the mitotic X 
chromosome really displays lower MeCP2 enrichment than the autosomes, at least using live 
imaging (a simple transfection would suffice in my opinion) but a mitotic ChIP-seq would be ideal. 
Then, the observation of high H3K9me3 along the mitotic chromosomal arms in MeCP2 KO cells is 
outstanding and should definitely be supported by a ChIP-seq. 




Reviewer #2 (Remarks to the Author): 

In this manuscript, Djeghloul and colleagues aim to identify proteins that convey epigenetic 
information through cell division. The authors isolated mitotic chromosomes using FACS from 
embryonic stem cells (ESCs) pharmacologically synchronized to mitosis and performed quantitative 
LC-MS/MS to detect proteins that are either enriched or depleted from mitotic chromosomes. The 
authors emphasize that they examine the proteome of the non-crosslinked chromosomes, thus 
avoiding fixation bias. In total, 614 proteins are found to be enriched on mitotic DNA. GO analysis 
of enriched proteins shows that these proteins have established roles in chromatin organization, 
DNA packaging, and chromosome architecture. The authors then focuses on the proteins involved 
in transcriptional repression and maintenance of heterochromatin. Using the Dnmt1,3a,3b -/- and 
Eed-/- ESCs that abolish the DNA methylation and H3K27me3 respectively, the authors show that 
loss of these repressive marks results in decompaction of mitotic chromosomes. The authors also 
show that loss of methyl binding protein Mecp2 leads to decompaction of mitotic DNA; however, 
the effect is limited to chromosomes that Mecp2 is binding to. Next, using the same approach for 
mitotic chromosome isolation as for their proteomic analysis, the authors demonstrate that mitotic 
ESC chromosomes are larger than their equivalents isolated from differentiated cells. In the final 
part of the manuscript, the authors show the mitotic chromosomes isolated using their approach 
are responsive to biological stimuli. Using a cleavable form of Rad21 (a component of the cohesin 
complex) in pre-B cells, the authors show that their purified native mitotic chromosomes are 
susceptible for cohesin cleavage, that results in decondensation of mitotic DNA. 

While the topic of the maintenance of epigenetic information through the mitosis is timely and 
important, the presented manuscript has several issues that question the results presented. 

1. The main concern with the presented manuscript is the method used to isolate native mitotic 
chromosomes. To this reviewer, the complex and multistage process of isolating mitotic DNA used 
by the authors, which includes polyamine-based chromosome release followed by DNA sorting, 
likely results in drastic changes of mitotic DNA that could affect the outcome of the study. Images 
of purified chromosomes resemble a blob (Figure 2-5 and S1-S3) do not look like metaphase 
chromosomes in cells or obtained from classical techniques such as the chromosomal spread. 
Furthermore, it is difficult to judge how this process that results in a distinct morphological change 
to chromosome structure affects the proteins that are bound to mitotic DNA. The authors need to 
validate the results obtained from the purified mitotic chromosomes using in vivo or in situ 
approaches. Furthermore, the subsequent experiments that showing changes in mitotic 
chromosome size or condensation should be validated either using FISH or by chromosomal 
spreads. 

2. Of note, this is not the first study that examines proteins bound to native mitotic chromosomes. 
In 2010, Ohta and colleagues performed proteomic analysis of mitotic chromosome proteins in 
DT40 cells. Surprisingly, this study is neither mentioned nor discussed in the presented 
manuscript. It is unclear how the current study advances findings presented in the 2010 study, 
particularly since the current study identified much fewer number of factors binding to mitotic 
chromosomes. Indeed, this may signify a limitation of the current protocol, which relies on 
extensive isolation process. 

3. Another issue is that in the current version the manuscript lacks focus. A large part of both 
introduction and discussion is dedicated to the maintenance of information through mitosis by 
transcription factors (TF). However, TFs are not the main focus of this manuscript and are only 
briefly mentioned in Figure 1. Instead, authors investigate the transcriptional repressors that are 
bound to mitotic chromosomes, which role is not established during mitosis. Authors suggest that 
these repressors are involved in the maintenance of DNA condensation during mitosis by 
measuring the size of their purified chromosomes. However, authors do not proceed any further to 
verify their observations in live cells, or to establish the impact of their observation on either cell 
division or maintenance of epigenetic information through mitosis. Even more, the possible 
relevance of their observations is not even adequately discussed. The results section focusing on 
Mecp2 is not properly introduced, and it is completely unclear why the authors decided to 
investigate the distribution of histone H3K9me3 along the mitotic chromosome. Again, there is no 
attempt to understand the functional relevance of this finding. 



Reviewer #3 (Remarks to the Author): 

This manuscript identifies factors enriched on mitotic chromosomes, with some follow up 
experiment to show that key repressive factors condense chromatin. Further, sizes of 
chromosomes appear dependent on differentiation state. This is an interesting manuscript that will 
be relevant for many chromatin researchers. I have some comments: 

When factors are found to bind mitotic chromatin this does not mean they are associated with 
their cognate binding sites (they may bind mitotic chromatin, but not sequence-specifically, so that 
would not be a bookmarking interaction necessarily). Imaging and proteomics will also observe 
such factors that are non-sequence-specifically bound to chromatin (nicely reviewed in Raccaud 
and Suter FEBS Lett 2018). It may be good to include mention of this in the manuscript. 

For the ESC proteomics: It seems most cells are indeed in G2 or M. The authors need to show how 
many cells are actually in metaphase. It is possible that the entire population has only few 
metaphase cells and many G2 cells. When the authors then purify mitotic chromosomes from this 
culture they will selectively extract these from the subpopulation of metaphase cells, and not from 
the non-metaphase cells. However, the lysate will have both metaphase and the many G2 (non-
mitotic) cells as well (i.e. the B-cell lysate in S5b appears mostly G2, not M as the Hi-C map is 
dominated by interphase features). This could severely affect the proteomic comparison between 
mitotic chromosomes and the lysates. Thus, the authors must show that most cells are no only 
having a G2 DNA content but are in (pro-)metaphase. The authors report finding known 
chromosome associated factors (Esrrb, Sox2), while finding that proteins known not to associate in 
G2 or M (MCM proteins) are depleted. Note that the latter depletion is not evidence that the lysate 
are good controls for metaphase chromosomes (see point above), as MCM proteins dissociate 
already in G2. 

Do the Hoechst and Chromomycin A3 dyes affect protein-DNA interactions? 

Overall the purified chromosomes look very odd. This is not what typical prometaphase 
chromosomes look like at all. Is this due to the isolation procedure? 

The authors measured size of chromosomes and centromeres separately. Did the centromere 
change volume change particularly when Dnmt1,3a,3b, MeCP2 or Eed were deleted? Any specific 
effects at centromeres as compared to arms? 
For the cohesin cleavage experiments: Maybe the centromeres are mostly responsible for size 
increase, given that cohesin presence on arms is very low. The authors mention that they measure 
size of centromeres separately. So can the authors report the changes in size of centromeres and 
the extent to which any such changes can explain the change in whole chromosome size? 

The authors state: "native metaphase chromosomes purified by FACS, remain responsive to 
appropriate biological cues". TEV cleavage of a protein is hardly an appropriate biological cue. 

The different size of mitotic chromosomes in ESCs vs differentiated cells is very interesting. I think 
something similar has been observed in published work from the Heald lab in Xenopus. 

Other comments: 

Can the authors provide a more detailed explanation about the Mitotic Lysate as controle was 
generated. No or few details mentioned in the Material & Methods. 

Can the authors compare proteins are enriched on mitotic chromosomes between cell lines? This 
may explain size differences, and also serve as another threshold for finding significant proteins. 

It is a bit puzzling that cohesin is statistically significantly enriched on mitotic chromosomes given 
that bulk cohesin is unloaded from chromatin in prophase. 

For figure 2 and 3 I would suggest doing normal Mitotic spreads as control, especially for the 
H3K9me3. 

For the chromosome size differences between cell lines, cells have to be arrested with similar time 
in colcemid as this affects chromosome size. (ESC and pre-B for 6h vs fibro and cardio for 12h). 

For Figure 2 and 3 mitotic spreads might tell a lot more about chromosome size as control. This 
could be used to compare fixed vs unfixed cells, yet ratios should not change. 

Supplementary figure 4, except for pre-B cells the size (uM) does not change from G1 to G2. PI 
intensity is normalized by the cell size so shouldn’t the PI intensity double. This seems not to be 
the case for 3 of 4 cells. 

Last, in the methods and materials all the centrifuge speeds are written as rpm without mentioning 
the centrifuge type, so maybe change it to rcf for reproducibility. 



Reviewer #4 (Remarks to the Author): 

In this manuscript, Fisher and colleagues study the factors associated with chromosomes. They 
sorted mitotic chromosomes using flow cytometry and analyzed the proteins co-enriched with the 
chromosomes. Among enriched proteins many of the expected transcription factors, chromatin 
modifiers, etc. were found as a positive controls for the experiment. The MS experiments are well 
described, the data is of high quality and acquired using robust protocols and high standards. 

A few minor points to be addressed: 

The authors discuss the mitotic bookmarking problem. It is a challenging problem and requires a 
multi-angle approach to assign or score functional relevance of proteins to mitotic chromosomes. 
For instance as described in PMID: 24561620. It is good that the manuscript does not make strong 
claims that proteins enriched in a proteomics screen must be relevant to chromosome biology. 

Especially, since it depends on an arbitrary decision how to define significantly enriched proteins. 
The MaxLFQ algorithm is based on the assumption that most of the proteins do not change 
between the samples. However, in the study more than 60% of proteins identified in chromosomes 
enriched sample are noted as being significantly enriched or depleted. Does this have an effect on 
how to select the criteria for significantly regulated proteins? 

In Figure 1c-f the x axis is labeled as “relative intensity”. The x axis shows a difference between 
two conditions in log2 space or an intensity ratio in a linier space typically noted as enrichment. 
It looks like that Figures 1d-f are derived from figure 1c by removing most of the points, but the 
significance cut-off of 1% FDR was not adjusted for individual subsets. One way to emphasize 
selected proteins would be to make all other proteins marked using transparent symbols and keep 
the same FRD cut-off for all the plots. 

Could you please provide the supplementary figure showing pairwise comparison between 
biological replicas including correlation coefficients. That would help to assess the reproducibility of 
the proteomics measurements. 
In the supplementary methods description could you please describe the steps preformed using 
Perseus software and which version of the software was used. 
Have you identified any proteins in chromosome enriched fraction which were not found in the 
total lysate? Is there a lot of proteins in the lysate identified only using match between runs 
functionality? 

Have you identified any chromosome associated proteins which are annotated as solely 
cytoplasmic proteins? That would help to assess the robustness of the enrichment protocol. 

A hierarchical clustering is a nice representation of the proteomics data. It would serve a similar 
function as figures 1c-f, but would allow to explore all potentially similarly behaving protein 
groups. For example see the publication PMID: 29208753. Please consider adding this.