The EMBO Journal Peer Review Process File - EMBO-2014-89957
Manuscript EMBO-2014-89957
Functional screen reveals essential roles of miR-27a/24 in
differentiation of embryonic stem cells
Yanni Ma, Nan Yao, Guang Liu, Lei Dong, Yufang Liu, Meili Zhang, Fang Wang, Bin Wang,
Xueju Wei, He Dong, Lanlan Wang, Shaowei Ji, Junwu Zhang, Yangming Wang, Yue Huang and
Jia Yu
Corresponding author: Jia Yu, Institute of Basic Medical Sciences, Chinese Academy of Medical
Sciences and Peking Union Medical College
Review timeline: Submission date:
12 February 2014
Editorial Decision:
18 February 2014
Transfer to EMBOReports:
20 February 2014
Editorial Decision:
17 March 2014
Author inquiry to The EMBO Journal
13 June 2014
Additional Editorial Correspondence by The EMBO Journal :
23 June 2014
Additional author correspondence to EMBO Reports and The EMBO Journal 25 June 2014
Resubmission :
01 September 2014
Editorial Decision
06 October 2014
Revision received:
28 October 2014
Accepted:
29 October 2014
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.)
Editors: Thomas Schwarz Romond and Esther Schnapp
1st Editorial Decision
18 February 2014
Thank you very much for your interest in The EMBO Journal and submitting your study for
consideration to our editorial office. I assessed the manuscript now in detail and discussed potential
suitability for The EMBO Journal with all members of our editorial team.
We appreciate the integrating approach to identify and characterize differentiation-promoting
miRNA's by a relative comprehensive set of functional assays. We also realize that at least 7
candidates are indeed corroborated in wildtype ESCs and linked to direct regulation of known
regulators of the pluripotency network. Focusing on one specific cluster, regulation downstream of
myc and improved somatic reprogramming upon depletion complement this certainly valuable,
though rather more resource-type set of experiments.
Though appreciating potential interest for the immediate field, significant conceptual notions on the
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role of miRNAs in driving ESC-differentiation (that includes some of your own previous studies) as
well as at the level of myc-regulation and principle involvement in efficiency of reprogramming
prohibit general enthusiasm at least at our more general and conceptual-oriented title.
Fully aware of the inherent interest as a resource for the stem cell and/or miRNA communities, I
took the liberty to inquire with our sister title EMBO Reports that could serve as alternative venue
for probably efficient dissemination of your findings. I am thus happy to inform you that their
scientific editor Esther Schnapp would be delighted to arrange for external peer-review.
I do hope that this presents a suitable and viable alternative for potential presentation of your data.
Please do not hesitate to contact Esther in case of any further questions ([email protected]).
I am sorry that I am unable to reach a more positive conclusion from the perspective of The EMBO
Journal, but I hope that the negative outcome on this occasion does not prevent you from supporting
and considering The EMBO Journal for publication of future studies.
Transfer to EMBO Reports
Editorial Decision by EMBO Reports
20 February 2014
17 March 2014
Thank you for the submission of your manuscript to EMBO reports. We have now received the full
set of referee reports that is copied below.
As you will see, the referees acknowledge that the findings are interesting and generally well
supported by the data. However, they also feel that some aspects of the study need to be
strengthened. The most important concerns that need to be addressed are whether the miR-23/24/27
cluster has an endogenous role in mESC differentiation/self-renewal (as mentioned by referee 2),
and the regulation of c-Myc expression by miR-27a/24 (noted by referee 1). Referee 2 further
remarks that it should be investigated which lineages the miRNA cluster induces, and in which
specific lineages these miRNAs are upregulated. All referees further pinpoint missing controls,
statistical analyses and missing details on experimental procedures and in figure legends, which
need to be provided. It is not strictly required for publication of the manuscript by EMBO reports
that you address the concerns raised by referee 1 in point 5 and by referee 3 in point 7.
Given these constructive comments, we would like to invite you to revise your manuscript with the
understanding that the referee concerns (as mentioned above and in their reports) must be fully
addressed and their suggestions taken on board. Acceptance of the manuscript will depend on a
positive outcome of a second round of review. It is EMBO reports policy to allow a single round of
revision only and acceptance or rejection of the manuscript will therefore depend on the
completeness of your responses included in the next, final version of the manuscript.
I look forward to seeing a revised version of your manuscript when it is ready. Please let me know if
you have any further questions or comments regarding the revision.
REFEREE REPORTS:
Referee #1:
The report by Ma Y. et al., uses a bioinformatic analysis to select microRNAs involved in
embryonic stem cell (ESC) differentiation. After an initial selection of 40miRNAs, the authors
narrow down their selection using a number of criteria to identify two miRNAs (miR-27a and miR24) that they further characterized. The authors then use a variety of approaches to show a potential
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direct regulation of several pluripotency factors and/or factors critical for the biology of ESC by
these miRs. They further show that c-Myc represses a cluster of miRNA containing miR-27a and
miR-24. The authors also provide interesting data supporting a function for these miRNAs in
reprogramming. Although there are some interesting new findings, this report contains several
inconsistencies and overstatements that need to be addressed.
My comments are as follow:
In the abstract the authors claim that "these factors can biologically repress Myc". However their
data does not support this claim. In general their data on these miRNAs regulation of c-Myc is
inconsistent.
1) In line 145 the authors forgot to mention the gene expression data on Sup. Fig. S4.
2) Figures 4 and 5: the authors should perform WB for miR-27a mimic-ESC for c-Myc, Nanog,
Oct4 and Smad4 to show that the silencing effect on these factors is specific to miR-24, and viceversa.
3) Figures 4 and 5 (a and b panels) are a bit redundant; the data should be re-arranged and the effect
of miR-27a and miR-24 overexpression in Dgcr-null or WT-ESC in silencing pluripotency factors
should be presented together.
4) In table S4 it should be specified which are the units for the values
5) The data on the seven miRNAs identified to induce ESC differentiation is an important finding
since as the authors state they do not seem to be negatively regulated by other miRNAs. The authors
should flesh this out by co-overexpressing miRNAs from the ESCC family with each of these
differentiation-inducing miRs to corroborate their hypothesis.
The report clearly identifies a repressive role of c-Myc on the expression of miR-23aò27aò24
cluster. However, the existence of a negative feedback loop of miR-27a and miR-24 to silence cMyc is an overstatement. In general the data is confusing, there are discrepancies that need to be
addressed.
(a) the heat map of Figure 2c showed downregulation of cMyc in ESC overexpressing all three
members of the cluster miR-23a, miR-27a and miR-24.
(b) Data in Suplemmentary Fig S4 displayed downregulation of c-Myc expression only in ESC
overexpressing miR-27a and miR-23a, but not the ones overexpressing miR-24.
(c) Bioinformatics analysis found only miR-24 binding sites in c-Myc coding sequence and reduced
c-Myc protein levels were shown only in miR-24 overexpressing ESC (Figure 5a and b), although
this was probably due to an indirect effect since Luciferase expression controlled by c-Myc CDS or
UTR was not affected in ESC-miR-24 mimic (Figure 5c).
6) In Fig 7 the authors should specify in the material and methods exactly how the endogenous and
exogenous OCT4, etc were distinguished.
Referee #2:
Review of manuscript by Ma et al. titled "Functional screen reveals essential roles of miR-27a/24 in
differentiation of embryonic stem cells"
Our knowledge of microRNAs that instruct mouse embryonic stem cells (mESCs) to exit
pluripotency and initiate differentiation remains somewhat underdeveloped to date. To identify
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novel microRNAs that fulfill such a function, Ma et al. individually overexpressed several dozen
miRNAs in DGCR8-/- mESCs (which lack endogenous miRNAs). The authors collectively
characterize two hits from their screen (miR-24 and miR-27a) whose ectopic induction can suppress
pluripotency factor expression and thereby drive mESCs towards differentiation. These
differentiation-inducing miRNAs are apparently downregulated by pluripotency factor c-Myc,
perhaps explaining why they are minimally expressed in mESCs. Reciprocally, depleting these
miRNAs in fibroblasts modestly enhances (2-fold) the subsequent generation of induced pluripotent
stem cells (iPSCs), functionally implying these miRNAs indeed attenuate pluripotency.
Strengths of the study
1. To nominate novel miRNAs that might initiate mESC differentiation, Ma and colleagues
identified miRNAs that were upregulated in differentiated lineages (fibroblasts and embryoid
bodies) over ESCs and that were 2) bioinformatically predicted to target pluripotency genes. With a
list of candidate microRNAs, Ma et al. individually overexpressed each of them in a large functional
screen in DGCR8-/- mESCs and subjected the resultant cells to multiple assays including colony
forming and replating assays, alkaline phosphatase (AP) staining, G1 phase accumulation tests and
real-time qPCR quantification of pluripotency markers to identify microRNAs that are functional
regulators of pluripotency. This systematic approach led them to focus on two microRNAs, miR-24
and miR-27a. Next, the authors continued to characterize their functional targets. Ma et al. found
these microRNAs repress pluripotency factors Oct4, Smad3, Smad4, Foxo1 and gp130, and that
they repress reprogramming.
2. One elegant aspect of their work is that the authors employed DGCR8-/- mESCs in their initial
screen, enabling a more robust readout of microRNA function in the absence of other microRNAs.
The authors then validated the microRNA candidates in another wild type ESC line and observed
similar effects upon pluripotency factor protein expression but not in the AP colony-forming assay.
3. The detailed characterization of the targets of miR24 and miR-27a - including multiple methods,
including protein analysis, luciferase assays, mutagenesis assays, ribonucleoprotein
immunoprecipitation - show consistently that miR-27a and miR-24 respectively target Foxo1,
Smad3, gp130, and Oct4. miR-24 reduces Oct4 3'UTR reporter constructs variably between ~2040%. Inhibition of these microRNAs appears to show an opposite trend for most of the pluripotency
factors, except for Smad3 and Smad4.
4. Finally, the figures are overall well organized and well annotated.
Suggestions for the authors
The most impressive part of this work is the functional miRNA overexpression screen in DGCR8-/mESCs (that lack any endogenous miRNAs), yet this has two intrinsic limitations.
1. Firstly, although the initial screen revealed miRNAs that potently suppressed self-renewal in the
'sensitized' background of DGCR8-/- mESCs (primarily miR-24, although miR-23a to a lesser
extent), when the authors moved to wild-type mESCs, much more modest effects were evidenced:
miR-24 had a modest phenotype and miR-23a failed to show any effect (Fig. 4a,b). This brings into
question the physiologic significance of these miRNAs. Although the authors have noted this in the
manuscript, perhaps they could discuss this more clearly.
2. Secondly, the authors convincingly show (at least in the DGCR8-/- background) that ectopic
miRNA overexpression annuls self-renewal, but this could potentially be attributed to high (nonphysiological) levels of miRNA overexpression. The authors clearly have effective miRNA inhibitor
reagents (Fig. 6) and therefore it would be important to establish whether miR-23/24/27 have any
endogenous role in mESC differentiation. That is, whether blockade of miR-23/24/27 (individually
or in combination) within wild-type mESCs can delay the onset of differentiation and safeguard
self-renewal to some extent. Indeed if for example miR-24 directly represses Oct4 expression, then
its inhibition might be anticipated to enhance mESC self-renewal. In any case, inhibition of these
microRNAs in wild-type mESCs would uncover their function in a physiological context and enable
a better assessment of their importance in the midst of other microRNAs.
4. The novel contribution of this study is the identification of novel differentiation-inducing
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miRNAs. Therefore it should be clarified whether these microRNAs have any early role in
mediating early cell fate decisions by more clearly parsing the lineage outcomes that are specifically
induced upon ectopic induction of these miRNAs (the authors noted weak upregulation of some
differentiation markers, but not with sufficient precision to identify the exact lineage outcomes).
Along this line of inquiry, the authors assayed the expression of ectoderm, mesoderm,
trophectoderm and endoderm markers in Figure 2c, yet the markers used are not entirely specific to
the lineage they were grouped under. For example, Fgf5 is expressed in epiblast stem cells,
Brachyury is expressed in the primitive streak, Hand1 is expressed not only in mesoderm but also
trophectoderm and lastly Foxa2 is expressed not only in endoderm but also in axial mesoderm.
Therefore, the authors could keep this in mind
while categorizing the markers into specific groups.
5. The effects of miR-23/24/27 are pronounced in DGCR8-/-mESCs but less so in wild-type ESCs
and in the latter context are largely examined by Western Blot (Fig. 4b), it would be useful to
replicate the experiments shown in Figure 4c and Figure 4e in wild type mESCs.
6. Although EB- and RA-based differentiation was used to identify the microRNAs that were
differentially expressed between pluripotent and differentiated states, these differentiation methods
generate multiple heterogeneous cell populations. While these experiments imply these miRNAs are
generally upregulated during 'differentiation', the authors should more precisely ascertain the
specific lineages (e.g., epiblast; primitive streak or ectoderm) in which these miRNAs are
upregulated (because after all, this study revolves around the role of the microRNAs on
differentiation).
Summary
Overall, the authors have done a thorough and careful job in parsing the role of miR-27a and miR24 and showed convincingly their role in targeting pluripotency genes. Therefore the large volume
of data in this study should further our understanding of pluripotency, differentiation and
microRNAs by identifying novel differentiation-inducing miRNAs. However, I believe the authors
should clarify whether these miRNAs are endogenously required during mESC differentiation to
silence self-renewal and execute differentiation (see above), amongst other points listed above and
some minor comments below, and I think addressing these concerns could further improve their
already impressive manuscript.
Minor comments
1. The distinction between wild type or DGCR8 mESCs should be made in the Figures 1, 2, 4, 5 and
their legend to clarify what cell type was used in the experiment. For example, indicate in figures 2a,
c, d, and 1d whether wild type or DGCR8-/- mESCs was used. In figure 3a, indicate 'wild type' in
the Figure.
2. Page 2, line 27 - The phrase "pre-selected collection of 40 microRNAs in Dgcr-8-null ESCs" in
the abstract needs to be clarified by including a brief statement of the criteria used to select these 40
microRNAs.
3. Page 2, line 51 - "ESCs lacking of key enzymes" with "ESCs lacking key enzymes"
4. Figure 7e - correct miR-241# to miR-24 #1.
5. Include the detail that regulation of targets by miRNA is direct.
6. Page 7, line 147 "microRNA mimic treated ESCs" to "microRNA mimic-treated ESCs".
7. Mention whether the rank from Figure 2e is based on the microarray data or real-time PCR
validation.
8. Include negative control for Figure 5g. - a gene that is not regulated by miR-24 and miR-27a.
9. Figure legend of Figure 2c may be clarified and explained. For example, is this gene expression
profile after microRNA overexpression in DGCR8-/- mESCs?
10. For Fig. 4c,e it would be useful to annotate those subpanels in the figure itself as being
conducted in DGCR8-/- mESCs, given that wild-type mESCs are also discussed in the
accompanying text
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Referee #3:
The manuscript by Ma et al. presents interestesting findings of several miRNAs capable of
suppressing ESC self-renewal and/or inducing their differentiation, and the conclusions are
supported by several different approaches. Particular focus is given to the miRNAs of the miR23a/miR-27a/miR-24 cluster and knowledge about their role in ESC differentiation might benefit
future work in field of cellular reprogramming as shown by the authors. The findings are of general
interest and manuscript for most parts well written. However, more details should be provided for
certain technical aspects, such as the exact Dgcr8-/- cell line used, and throughout the manuscript
the figure legends should be more detailed. Also, more clarafication and analysis would be needed
especially on selection of the miR-23a/miR-27a/miR-24 cluster and on the experiments about the
transcriptional regulation of this cluster by c-Myc.
More detailed major comments are listed below:
1) Some bioinformatic analysis require more details in Materials&Methods or Figure Legends. For
example, from TargetScan predictions in Supplementary Table S3, which exact tool, which version,
and which settings were considered for selecting binding sites (seems all possible were included).
Also, which RNA structure prediction software was used for miR-23a/miR-27a/miR-24 cluster
structure in Fig3B? No reference/link is provided. What message do the authors want to convey with
this panel (3B)? Please explain the details of this (and most other) figure legends better.
2) The Dgcr8-deficient ES cell line, which is a central model system of this manuscript, is first
mentioned at page 6. However, no reference to the origin of the cell line is provided. This should be
included in minimum in the Materials and Methods. Have the authors received the cell line from
somewhere or created it themselves? If latter, all details of cell line and the knock-out should be
provided.
3) Fig 2C: This is another example of a figure panel that lacks sufficient details. Equally the details
are missing from the corresponding Supplementary Table S6. What values does the color bar or the
numbers in the table represent (fold change, linear or log scale, relative to what)? At least in the
supplementary table, also statistical significance of these changes should be provided, if the data are
based on biological triplicates.
4) Page 8, "Grading and Scoring of screening results": This chapter is written somewhat vaguely and
description of Fig 2E should be more detailed given its central role for the manuscript (including
references to previous figures).
5) Page 9: Logic for selection of miR-23a/miR-27a/miR-24 cluster for further studies should be
better clarified. Why miR-23b/miR-27b/miR-24 cluster was not studied further although it also
expresses miR-24 and miR-27b has the same seed as miR-27a (and therefore presumably shares
many of its targets)? Is miR-23b/miR-27b/miR-24 cluster expressed with similar or different
patterns and what is its relative contribution to the total expression levels in different tissues,
including ESCs?
6) Fig 5C: what is the positive control used in the reporter gene assay? Figure should be consistent
with legend which should be consistent with details from materials and methods.
7) Fig 6C: What are the genomic coordinates and sequence of the miR-23a/miR-27a/miR-24 cluster
promoter and transcription start site considered for the experiments? Is the use of this transcription
start site in ESCs supported by existing publically available epigenomic data such as H3K4me3 or
H3K36me3 enrichments (e.g ENCODE)? Are there alternative transcription sites? Is the binding of
c-Myc to this transcription start site supported by existing publically available ChIP-Seq data for cMyc binding in ESCs?
8) Fig 6D: Please indicate the locus of the used negative control region.
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Additional minor comments:
1) Please avoid using unspecifying abbreviations (like etc.) in the abstract.
2) there should always be space between numbers and units. For example, "100 nM" instead of
"100nM".
Author Enquiry to the EMBO Journal
13 June 2014
A few months ago, we had submitted a manuscript entitled °∞Functional screen reveals essential
roles of miR-27a/24 in differentiation of embryonic stem cell°± (EMBOJ-2014-88205) for your
consideration to publish on The EMBO Journal. In the manuscript, we reported the identification of
a novel class of ES cell differentiation-promoting miRNAs and clarified the important role of miR27a/24 in controlling mESC state and iPSC generation, also the underlying mechanisms. The
manuscript was later transferred to EMBO Reports following your kindly recommendation. After
peer-review, all the reviewers expressed highly positive statements on the manuscript and also
provided some helpful comments and suggestions. We have revised the manuscript carefully, and
moreover, we have labored to define the physiological and endogenous role of the miR-23a cluster
in mESC differentiation. Taking advantage of the newly-developed CRISPR/Cas9 genome editing
system, we generated several miR-23a cluster and/ormiR-23b cluster bi-allelic knockout ES cell
clones and found that the double-knockout cells displayed differentiation deficiency during
embryoid body (EB) formation. The results greatly advanced the significance of our findings for the
following reasons: i. miR-23a/b clusters were improved to be absolutely necessary for mESC
differentiation which was different from the redundant roles of the most miRNAs in many biological
processes. This also suggested their indispensable roles in ontogenesis. ii. As we know, this is the
first study to identify miRNAs function in ES cells using CRISPR/Cas9 technology. Our study
reveals a feasible and efficient approach for bi-allelicablation of two miRNA clusters
simultaneously in ESCs anddemonstrates its usefulness in elucidating miRNA function. iii.
Considering the prevalent redundancy in the miRNA family, our study is a good sample showing
how to decipher the function of miRNAs in biological processes. It should be of broad interest to the
readership of The EMBO Journal.
In consideration of the positive statements from the reviewers and the important new findings, may I
take the liberty of asking for your reconsideration of the manuscript for publication in The EMBO
Journal? In addition, the manuscript text and figures largely exceeds the limits of EMBO Reports.
We are afraid the integrity and significance of our findings will be declined if the manuscript is
shortened to fit the requirement.
The new findings and the comments of the reviewers are provided in the attachments.
We look forward to your response.
Additional Editorial Correspondence by The EMBO Journal
23 June 2014
I now found the time to look at the formally submitted revised version to our sister title
EMBOreports and discussed potential course of action.
I am very much impressed by what you already achieved. Governed by your proposal to finalize the
functional aanlyses (including careful examination and inclusion of the differentiation/terrratoma
data from the Crispr/Cas-mediated miRNA-deletion, and conditioned on the satisfactory outcome of
such further reaching results, I would be willing to reconsider an amended and appropriately drafted
full-article format manuscript for publication in The EMBO Journal.
Please realize that I would also be willing to engage some of the referees originally selected for the
assessment of your work at EMBOreports, but I can at this stage not formally predict/determine the
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EVENTUAL OUTCOME of such a final evaluation.
Please be assured that I would be overall rather perceptive to the idea of publishing the paper in The
EMBO Journal, given the recent progress and significant technological/functional development the
study seems to offer.
If this appears of interest to you, I kindly ask you to get in touch with the scientific editor at
EMBOreports (I cc Esther Schnapp and Nonia Pariente on this occasion). Only upon your formal
request they would put the process on hold/withdraw the paper to subsequently mediate transfer of
your future AND further revised study for consideration at The EMBO Journal.
I do look forward to learn about your position/decision how to proceed and will act accordingly.
Additional author correspondence to The EMBO Journal and EMBO Reports
25 June 2014
We are glad to receive your letter and appreciate your positive comments and anticipation to our
current/future work.
Based on your suggestions, we have emailed Dr. Esther Schnapp to formally withdraw the paper
and request a transfer for our revised manuscript entitled °∞Functional screen reveals essential roles
of miR-27a/24 in differentiation of embryonic stem cell°± (EMBOR-2014-38665V1) from The
EMBO Reports to The EMBO Journal.
In the meantime, we would like to ask for another 2-3 months to complete the further experiments,
which we have described to you in our previous emails, to carefully assess the differentiation
capacity of the miRNA cluster-knockout ES cells. We believe the in vivo data will extremely
strengthen our findings and provide significant technological/functional development in stem cells
and/or miRNA areas.
We will then provide an amended and appropriately drafted full-article for your consideration to
publish in The EMBO Journal. Thank you very much.
Resubmission
01 September 2014
We have submitted our revised manuscript, entitled °∞Functional screen reveals essential roles of
miR-27a/24 in differentiation of embryonic stem cell°± (Transferred from EMBO reports, previous
number is EMBOR-2014-38665V1) to The EMBO Journal.
We appreciate the reasonable and helpful criticisms and suggestions from the reviewers and have
revised our paper according to the suggestions by: (i) incorporating additional data to answer some
of the points and to strengthen our conclusions; (ii) clarifying some areas that were confusing; and
(iii) adding additional methodological details and references. A point-by-point response is attached.
More importantly, we have designed the additional experiments to assess the differentiation capacity
of the CRISPR/Cas9 mediated bi-allelic miRNA-23~27~24 clusters knockout ESC clones in vivo
and directed to cardiomyocytes in vitro. In brief, the double knockout ESCs exhibit serious
deficiency in cardiomyocyte differentiation and the generated teratomas possess undifferentiated
regions indicative with Oct4 expression and much less mesoderm cells. The results greatly advanced
the significance of our findings for the following reasons: (i) miR-23a/b cluster was required for
mESC differentiation, which was different from the redundant roles of the most miRNAs in many
biological processes. (ii) Our study reveals a feasible and efficient approach for bi-allelic miRNA
ablation in ESCs and demonstrates its usefulness in elucidating miRNA function, which may be the
first study to identify miRNA function using CRISPR/Cas9; (iii) Considering the prevalent
redundancy in the miRNA family, our study is a good example showing how to decipher the
function of miRNAs in biological processes.
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We are grateful to all of the referees for their thoughtful and constructive suggestions, as these
suggestions have strengthened this manuscript. Since we have complied with virtually all of the
suggestions made by the three referees, we hope that you will now deem this paper acceptable for
publication in The EMBO Journal. As indicated by the positive testimonies of the reviewers, this
paper represents an important contribution to the area of microRNAs and embryonic stem cells, and
we are eager to see it in print.
In addition, the revised MS includes two supplementary movies, which is too big to be uploaded
online. So, we will send a FedEx to you soon. Besides, when the supplementary tables in excel are
automatically converted into PDF format during submission, format of table S7, S8 and S10
changed a little. Does this affect the peer-review process and what should we do about this?
Point-by-Point-Response:
Response to Referee #1:
The report by Ma Y. et al., uses a bioinformatic analysis to select microRNAs involved in
embryonic stem cell (ESC) differentiation. After an initial selection of 40 miRNAs, the authors
narrow down their selection using a number of criteria to identify two miRNAs (miR-27a and miR24) that they further characterized. The authors then use a variety of approaches to show a potential
direct regulation of several pluripotency factors and/or factors critical for the biology of ESC by
these miRs. They further show that c-Myc represses a cluster of miRNA containing miR-27a and
miR-24. The authors also provide interesting data supporting a function for these miRNAs in
reprogramming. Although there are some interesting new findings, this report contains several
inconsistencies and overstatements that need to be addressed.
My comments are as follow:
In the abstract the authors claim that "these factors can biologically repress Myc". However their
data does not support this claim. In general their data on these miRNAs regulation of c-Myc is
inconsistent.
The report clearly identifies a repressive role of c-Myc on the expression of miR-23a˜27a˜24 cluster.
However, the existence of a negative feedback loop of miR-27a and miR-24 to silence c-Myc is an
overstatement. In general the data is confusing, there are discrepancies that need to be addressed.
We agree with the reviewer that we did not directly demonstrate the regulatory role of miR-27a/24
targets on c-Myc expression. However, their interactions in ESCs have been reported by other
investigators formerly. For example, Smad3, a target of miR-27a, has been reported to be able to
bind onto c-Myc gene and activate its expression [Loh YH, et al., 2006, Nature Genetics, 38: 43140]. In addition, knock down of Oct4, a target of miR-24, also can induce down-regulation of c-Myc
in ESCs [Brown S, et al. 2011, Stem Cells, 29:1176-1185]. So we deduce that the important
pluripotency-associated targets of miR-27a/24 may have positive effects on c-Myc, and their
suppression induced by miR-27a/24 might repress c-Myc expression, thus forming a negative
feedback loop. Considering the comments from the reviewer, we deleted the description of the
feedback loop from the abstract and discussed the speculation in the discussion when rewriting.
The inconsistence about the regulation of these miRNAs on c-Myc is explained as follows:
(a) The heat map of Figure 2c showed down-regulation of c-Myc in ESC overexpressing all three
members of the cluster miR-23a, miR-27a and miR-24.
(b) Data in Supplementary Fig S4 displayed downregulation of c-Myc expression only in ESC
overexpressing miR-27a and miR-23a, but not the ones overexpressing miR-24.
In the heat map of Figure 2C, black color represents the mRNA level of this gene is unchanged. So
we can see that overexpression of miR-27a and miR-23a decreases c-Myc mRNA level obviously, by
contrast, overexpression of miR-24 does not affect c-Myc mRNA level which is consistent with the
results shown in the Supplementary Fig S4 C.
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Figure 2C The heatmap representation of gene expression pattern in miRNA mimic-transfected
Dgcr8-/-ESCs; Figure S4C Relative expression of pluripotency-associated genes in miRNA mimictransfected Dgcr8-/-ESCs.
(c) Bioinformatics analysis found only miR-24 binding sites in c-Myc coding sequence and reduced
c-Myc protein levels were shown only in miR-24 overexpressing ESC (Figure 5a and b), although
this was probably due to an indirect effect since Luciferase expression controlled by c-Myc CDS or
UTR was not affected in ESC-miR-24 mimic (Figure 5c).
We agree with the reviewer that the regulation of miR-­‐24 on c-­‐Myc expression is an indirect post-­‐
transcriptional effect. MiR-­‐24 might regulate c-­‐Myc expression at post-­‐transcriptional level since miR-­‐24 reduced c-­‐Myc protein but had no effect on its mRNA. However, miR-­‐24 did not target c-­‐Myc directly, because the luciferase expression controlled by c-­‐Myc CDS or UTR was not affected by miR-­‐
24 mimic. So it is possible that the post-­‐transcriptional processing or translation of c-­‐Myc is modulated by the targets of miR-­‐24 in ESCs, which needs to be validated further. References:
Brown S, Teo A, Pauklin S, et al. Activin/Nodal signaling controls divergent transcriptional
networks in human embryonic stem cells and in endoderm progenitors. Stem Cells 2011; 29:11761185.
Loh YH, Wu Q, Chew JL, Vega VB, Zhang W, Chen X, Bourque G, George J, Leong B, Liu J, Wong KY, Sung KW, Lee CW, Zhao XD, Chiu KP, Lipovich L, Kuznetsov VA, Robson P, Stanton LW, Wei CL, Ruan Y, Lim B, Ng HH. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet. 2006; 38(4):431-­‐40. 1) In line 145 the authors forgot to mention the gene expression data on Sup. Fig. S4.
The Sup. Fig. S4 is not the gene expression data but the ranking of the miRNAs based on their
ability to silence ESC self-renewal program, and include the data from colony formation assay, AP
staining, cell cycle, gene expression pattern and Oct4 staining.
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2) Figures 4 and 5: the authors should perform WB for miR-27a mimic-ESC for c-Myc, Nanog,
Oct4 and Smad4 to show that the silencing effect on these factors is specific to miR-24, and viceversa.
Although miR-24 but not miR-27a targets Oct4 and Smad4, miR-27a can also induce the changed
expression of Oct4 or Smad4 possibly through promoting the differentiation of ESCs. So the
silencing effect on Oct4 and Smad4 is possibly not specific to miR-24 and vice-versa. Actually, it is
not enough to determine that a microRNA targets a gene just according to the decrease of protein
level of its target in the microRNA overexpressing cells, as microRNAs can down-regulate some
non-targeting genes’ expression indirectly. The further reporter assays of the regulatory sequence
containing the microRNA binding site and the mutated site are necessary. In our experiment, all the
results of western blot, reporter assay and Ago2 RIP indicated that Foxo1, gp130 and Smad3 were
direct targets of miR-27a, Oct4 and Smad4 were direct targets of miR-24. So we think that detection
of the protein level of the targets of miR-24 or miR-27a in miR-27a or miR-24 mimic treated-ESCs is
of little significance for drawing the conclusion. The previous Figure 5B has been placed at Figure
4B in the revised manuscript.
3) Figures 4 and 5 (a and b panels) are a bit redundant; the data should be re-arranged and the effect
of miR-27a and miR-24 overexpression in Dgcr-null or WT-ESC in silencing pluripotency factors
should be presented together.
The panels have been combined together in the revised manuscript as Figure 3G.
4) In table S4 it should be specified which are the units for the values
We appreciate the suggestion of the reviewer and indicate the units for the values in table S4. The
values in table S4 are the average of the colony number of three repeats and are reserved as round
numbers in the revised manuscript.
5) The data on the seven miRNAs identified to induce ESC differentiation is an important finding
since as the authors state they do not seem to be negatively regulated by other miRNAs. The authors
should flesh this out by co-overexpressing miRNAs from the ESCC family with each of these
differentiation-inducing miRs to corroborate their hypothesis.
As our study showed, the seven miRNAs can decrease AP activity and produced smaller or flat cell
colonies in wild-type ESCs, though their effect was not as dramatic as that in Dgcr8-/- ESCs. Our
results also revealed that the effects of these seven miRNAs were antagonized by ESC specific or
enriched miRNAs but were not neutralized completely. Therefore, it is critical to study the
antagonistic effect between the differentiation-associated miRNAs and ESCC miRNAs. Our works
wish to identify novel differentiation-associated miRNAs through a screening different with other
studies in this area and tried to state the function and mechanism of miR-27a and 24 in ESCs. So we
think it is not necessary to study the antagonization of ESCC to them in our current work. But we
will explore this in our further study.
6) In Fig 7 the authors should specify in the material and methods exactly how the endogenous and
exogenous OCT4, etc were distinguished.
To distinguish the endogenous and exogenous Oct4, Sox2 and Klf4, the forward endogenous primers
were designed to be located in the 5’UTR region of the endogenous gene, whereas the reverse
exogenous primers were designed to be located in the vector. This has been indicated in Methods
and Materials.
Final Note: We have revised the manuscript according to the suggestions from the reviewer. In
addition, based on the suggestions from the editor and the other two reviewers, we also make the
following changes and supply some additional data in the revised manuscript:
1. We detected several self-renewal and differentiation markers using immunofluorescence in
miRNA transfected-wild-type ESCs to better understand the roles of these miRNAs in wild-type
ESCs (Fig 3I).
2. Excitingly, by using newly-developed CRISPR/Cas9technologywe generated miR-23a-27a-24-2
and/or miR-23b-27b-24-1 cluster knockout mESCs, which is more suitable in studying the
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endogenous and physiological role of the cluster in mESC differentiation substantially (Fig 6, 7 and
Supplementary Fig S7, 8).
3. We included negative control that was not regulated by miR-24 and miR-27a in Ago2-IP
experiments (Fig 4F, G).
All the major changes in the revised manuscript are marked as red characters.
Response to Referee #2:
Review of manuscript by Ma et al. titled "Functional screen reveals essential roles of miR-27a/24 in
differentiation of embryonic stem cells"
Our knowledge of microRNAs that instruct mouse embryonic stem cells (mESCs) to exit
pluripotency and initiate differentiation remains somewhat underdeveloped to date. To identify
novel microRNAs that fulfill such a function, Ma et al. individually overexpressed several dozen
miRNAs in DGCR8-/- mESCs (which lack endogenous miRNAs). The authors collectively
characterize two hits from their screen (miR-24 and miR-27a) whose ectopic induction can suppress
pluripotency factor expression and thereby drive mESCs towards differentiation. These
differentiation-inducing miRNAs are apparently downregulated by pluripotency factor c-Myc,
perhaps explaining why they are minimally expressed in mESCs. Reciprocally, depleting these
miRNAs in fibroblasts modestly enhances (2-fold) the subsequent generation of induced pluripotent
stem cells (iPSCs), functionally implying these miRNAs indeed attenuate pluripotency.
Strengths of the study
1. To nominate novel miRNAs that might initiate mESC differentiation, Ma and colleagues
identified miRNAs that were upregulated in differentiated lineages (fibroblasts and embryoid
bodies) over ESCs and that were 2) bioinformatically predicted to target pluripotency genes. With a
list of candidate microRNAs, Ma et al. individually overexpressed each of them in a large functional
screen in DGCR8-/- mESCs and subjected the resultant cells to multiple assays including colony
forming and replating assays, alkaline phosphatase (AP) staining, G1 phase accumulation tests and
real-time qPCR quantification of pluripotency markers to identify microRNAs that are functional
regulators of pluripotency. This systematic approach led them to focus on two microRNAs, miR-24
and miR-27a. Next, the authors continued to characterize their functional targets. Ma et al. found
these microRNAs repress pluripotency factors Oct4, Smad3, Smad4, Foxo1 and gp130, and that
they repress reprogramming.
2. One elegant aspect of their work is that the authors employed DGCR8-/- mESCs in their initial
screen, enabling a more robust readout of microRNA function in the absence of other microRNAs.
The authors then validated the microRNA candidates in another wild type ESC line and observed
similar effects upon pluripotency factor protein expression but not in the AP colony-forming assay.
3. The detailed characterization of the targets of miR24 and miR-27a - including multiple methods,
including protein analysis, luciferase assays, mutagenesis assays, ribonucleoprotein
immunoprecipitation - show consistently that miR-27a and miR-24 respectively target Foxo1,
Smad3, gp130, and Oct4. miR-24 reduces Oct4 3'UTR reporter constructs variably between ~2040%. Inhibition of these microRNAs appears to show an opposite trend for most of the pluripotency
factors, except for Smad3 and Smad4.
4. Finally, the figures are overall well organized and well annotated.
Suggestions for the authors
The most impressive part of this work is the functional miRNA overexpression screen in DGCR8-/mESCs (that lack any endogenous miRNAs), yet this has two intrinsic limitations.
1. Firstly, although the initial screen revealed miRNAs that potently suppressed self-renewal in the
'sensitized' background of DGCR8-/- mESCs (primarily miR-24, although miR-23a to a lesser
extent), when the authors moved to wild-type mESCs, much more modest effects were evidenced:
miR-24 had a modest phenotype and miR-23a failed to show any effect (Fig. 4a,b). This brings into
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question the physiologic significance of these miRNAs. Although the authors have noted this in the
manuscript, perhaps they could discuss this more clearly.
-­‐/-­‐
We appreciate the pertinent comments from the reviewer and agree with the reviewer that Dgcr8 mESC is more sensitive than the wild-­‐type mESC and some miRNAs exhibit silencing self-­‐renewal in -­‐/-­‐
Dgcr8 mESCs but have no visible effect in wild-­‐type mESCs. However, this does not indicate that these miRNAs are nonfunctional in ESCs and just suggests that it cannot be observed in wild-­‐type ESC through miRNA mimic over-­‐expression. Since genetic knockout of these miRNAs in mouse can reflect their physiologic roles better, we further performed CRISPR/Cas9 technology to knockout miR-­‐23/27/24 cluster to explore their physiologic roles in mESC differentiation. The detailed results and discussion has been supplemented in the revised manuscript. 2. Secondly, the authors convincingly show (at least in the DGCR8-/- background) that ectopic
miRNA overexpression annuls self-renewal, but this could potentially be attributed to high (nonphysiological) levels of miRNA overexpression. The authors clearly have effective miRNA inhibitor
reagents (Fig. 6) and therefore it would be important to establish whether miR-23/24/27 have any
endogenous role in mESC differentiation. That is, whether blockade of miR-23/24/27 (individually
or in combination) within wild-type mESCs can delay the onset of differentiation and safeguard
self-renewal to some extent. Indeed if for example miR-24 directly represses Oct4 expression, then
its inhibition might be anticipated to enhance mESC self-renewal. In any case, inhibition of these
microRNAs in wild-type mESCs would uncover their function in a physiological context and enable
a better assessment of their importance in the midst of other microRNAs.
According to the helpful suggestion from the reviewer, we examined whether blockade of miR23/24/27 within wild-type mESCs can delay the onset of differentiation and safeguard self-renewal.
To achieve blockade of the microRNAs better, we knocked out miR-23~27~24 cluster through
newly-developed CRISPR/Cas9 genome editing system instead of blocking them by miRNA
inhibitors. Five miRNAs (miR-23a, 23b, 27a, 27b, 24) constitute mouse miR-23~27~24 clusters,
which locate on two chromosomes as miR-23a~27a~24-2 and miR-23b~27b~24-1 cluster separately
(Fig 6A). So we generated miR-23a~27a~24-2 cluster (miR-23a cluster) and/or miR-23b~27b~24-1
cluster (miR-23b cluster) bi-allelic knockout V6.5 ESC clones. Both of the two clusters were biallelic knocked out in DKO ESC clone, while miR-23a cluster alone was ablated in KO ESC clone
(Fig 6B). All mature miR-23, miR-27 and miR-24 were undetectable in DKO ESC clone, while
mature miR-23b and miR-27b were normally expressed and miR-24 was reduced in KO ESC clone
(Fig 6C). Both KO and DKO ESCs were morphologically normal and expressed ESC specific
markers (Fig 6D-F).
To assess the differentiation capacity of the knockout ESCs, we cultured them as EBs. As shown
in Figure 7A, the up-regulation of Brachyury and Hand1 was delayed and was significantly lower
than wild-type levels in EBs derived from DKO cells. By contrast, the expression of Nestin and
Foxa2 was increased obviously, suggesting that the suppression of mesoderm lineages induced by
miR-23~27~24 cluster knockout might trigger the differentiation of other lineages. Meanwhile, the
decline of Oct4 during EB formation was also delayed in the DKO cells. In addition, the repression
of Brachyury and Hand1, as well as the promotion on the other lineage markers can also be
observed in EBs derived from KO cells, although the effect was weaker than that in DKO cells.
These results indicate that miR-23~27~24 clusters are required for silence of ESC self-renewal
program and formation of early mesoderm.
To further demonstrate their indispensable roles in an exact lineage differentiation, we conducted
cardiac differentiation of ESCs since mature miR-23/24/27 miRNAs were enriched in adult mouse
hearts (Fig 3F). On day 8 of differentiation (2 days after EB plating), about 40% of the plated wildtype ESC-derived EBs already showed spontaneous contraction. The percentage continued to
increase to 80%~90% at day 12 and this proportion was kept up till the final time point we
examined. In contrast, none of DKO ESC-derived EBs with spontaneous contraction was observed
during the whole differentiation period (Fig 7B). The morphology of DKO ESC-derived EBs was
also significantly different from that of wild-type ESCs, and showed many bulgy bubbles. KO ESCderived EBs showed distinctly decreased beating incidence and the beating clusters were usually
smaller than those in wild-type EBs (Fig 7B). These data correlated with the mRNA and protein
expression level of the cardiac marker genes. Real-time PCR revealed markedly lower expression of
Nkx2.5, Tbx5, α-MHC, β-MHC and cTnT in differentiating DKO and KO cultures compared with
wild-type ESC derivations. The peak expression of these cardiac markers was also delayed in DKO
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ESCs during differentiation (Fig 7C). Immunofluorescence analysis of differentiation cultures
revealed that V6.5 ESCs and KO ESCs-differentiated cells had well-organized Actinin, ANP and
Troponin I expression, which was more in V6.5 ESC cultures than that of KO ESC cultures. Instead,
DKO ESCs could not differentiate and form typical myofilament, with few expression of the three
cardiac markers (Fig 7D). These data support miR-23~27~24 clusters are absolutely required for
cardiac lineage differentiation of ESC in vitro and the roles of miR-23a cluster could be
compensated by miR-23b cluster partially.
Teratomas normally consist of a heterogeneous mix of differentiated cell types and usually are
used to test the pluripotency of stem cells in vivo. In accordance with the defects in EB and cardiac
differentiation, the teratomas produced with miR-23~27~24 clusters knockout ESCs grew slowly
and were significantly lighter than that derived from wild-type ESCs (Fig 7E, F). A certain
proportion of undifferentiated regions indicative with Oct4 expression were detected in DKO and
KO ESC derived teratomas, which were occasional in the wild-type derivations (Fig 7G). Moreover,
teratomas derived from DKO ESCs largely appeared ectoderm structures especially neuronal
rosette, while mesoderm cells such as adipocyte, cartilage and muscle cells were distinctly less than
that of wild-type ESCs (Fig 7H). Our results indicate that miR-23a and miR-23b cluster are
indispensable for ESC differentiation especially the mesoderm differentiation in vivo.
Figure 6 Bi-allelic double knockout of miR-23~27~24 clusters in ESCs by CRISPR/Cas9.
A. Gene location and sequence alignment of mouse miR-23~27~24 cluster miRNAs. Black box
indicates the seed region.
B. Genomic PCR of the specific fragments spanning miR-23~27~24 clusters. WT indicate wildtype ESCs; DKO indicate miR-23a~27a~24-2-/-, miR-23b~27b~24-1-/- ESCs; KO indicate
miR-23a~27a~24-2-/- ESCs.
C. Real-time PCR analysis of mature miR-23a/b, miR-27a/b and miR-24 expression in wild-type,
DKO and KO ESCs. Data are shown as mean + s.d. (n=3).
D. AP staining of wild-type and miR-23~27~24 cluster knockout ESCs.
E. Real-time PCR analysis of Oct4, Sox2 and Nanog expression in wild-type and miR-23~27~24
cluster knockout ESCs. Data are shown as mean + s.d. (n=3).
F. Oct4, Sox2 and Nanog immunofluorescence staining of wild-type and the knockout ESCs.
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Figure 7 miR-23~27~24 cluster are required for ESC differentiation.
A. Real-time PCR analysis of Oct4 and several lineage differentiation markers expression during
EB formation of wild-type and knockout ESCs. Data are shown as mean ± s.d. (n=3).
B. Beating EB incidence during cardiac differentiation of wild-type and knockout ESCs. Data are
shown as mean + s.d. (n=3). * indicates P-value < 0.05; ** indicates P-value < 0.01; ***
indicates P-value < 0.001.
C. Real-time PCR analysis of several cardiac markers expression during cardiac differentiation
of wild-type and knockout ESCs. Data are shown as mean ± s.d. (n=3).
D. Actinin, ANP and Troponin I immunofluorescence staining of differentiation cultures derived
from wild-type and knockout ESCs.
E. Volumes of teratomas at different days after injection. Data are shown as mean + s.d. (n=4). *
indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.
F. Weight of teratomas derived from wild-type and knockout ESCs. Data are shown as mean ±
s.d. ** indicates P-value < 0.01.
G. Oct4 immunohistochemistry staining of teratomas derived from wild-type and knockout ESCs.
Representative Oct4 positive regions are shown.
H. Hematoxylin and eosin staining of teratomas derived from wild-type and knockout ESCs.
Ectoderm, Mesoderm and Endoderm are marked with different color arrows.
4. The novel contribution of this study is the identification of novel differentiation-inducing
miRNAs. Therefore it should be clarified whether these microRNAs have any early role in
mediating early cell fate decisions by more clearly parsing the lineage outcomes that are specifically
induced upon ectopic induction of these miRNAs (the authors noted weak upregulation of some
differentiation markers, but not with sufficient precision to identify the exact lineage outcomes).
Along this line of inquiry, the authors assayed the expression of ectoderm, mesoderm,
trophectoderm and endoderm markers in Figure 2c, yet the markers used are not entirely specific to
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the lineage they were grouped under. For example, Fgf5 is expressed in epiblast stem cells,
Brachyury is expressed in the primitive streak, Hand1 is expressed not only in mesoderm but also
trophectoderm and lastly Foxa2 is expressed not only in endoderm but also in axial mesoderm.
Therefore, the authors could keep this in mindwhile categorizing the markers into specific groups.
We appreciate the helpful comments of the reviewer. The ambiguous categorization of germinal
layer markers has been deleted from Figure 2C.
In our study, we identified the miRNAs mainly based on their ability to silence ESC self-renewal.
The miRNAs chiefly exhibited suppressing self-renewal and the repression of self-renewal program
would result in spontaneous differentiation of mESCs. So we think the up-regulation of some
differentiation markers induced by ectopic induction of these miRNAs may be due to spontaneous
differentiation of mESCs but not exact lineage differentiation. It is difficult to achieve an exact
lineage outcome just through ectopic induction of a specific microRNA in ESCs. Although we did
not clearly parse the lineage outcomes induced upon miR-27/24, we revealed the essential roles of
miR-27/24 in mesoderm differentiation of ESC by generating and investigating miRNA-23~27~24
clusters knockout ESCs, which also provide a good example showing how to decipher the function
of miRNAs in biological processes.
5. The effects of miR-23/24/27 are pronounced in DGCR8-/-mESCs but less so in wild-type ESCs
and in the latter context are largely examined by Western Blot (Fig. 4b), it would be useful to
replicate the experiments shown in Figure 4c and Figure 4e in wild type mESCs.
According to the helpful suggestion from the reviewer, we detected several self-renewal and
differentiation markers using immunofluorescence in miRNA transfected-wild-type ESCs. As shown
in Fig. 3I, obviously decrease of Oct4 and Nanog, accompanied by elevation of Foxa2, Gata2 and
Nestin was observed in let-7c, miR-27a and miR-24 overexpressed wild-type ESCs. The results
indicate that miR-27a and miR-24 are critical negative-regulators of ESC self-renewal and
participate in the differentiation of ESC.
Figure 3I Oct4, Nanog, Foxa2, Gata2 and Nestin immunofluorescence of mimic transfected-wildtype ESCs.
6. Although EB- and RA-based differentiation was used to identify the microRNAs that were
differentially expressed between pluripotent and differentiated states, these differentiation methods
generate multiple heterogeneous cell populations. While these experiments imply these miRNAs are
generally upregulated during 'differentiation', the authors should more precisely ascertain the
specific lineages (e.g., epiblast; primitive streak or ectoderm) in which these miRNAs are
upregulated (because after all, this study revolves around the role of the microRNAs on
differentiation).
We appreciate the reasonable suggestions from the reviewer and agree that it is helpful to define the
specific lineages in which miR-23a cluster members are up-regulated. However, the specific lineage
stage during ESC early differentiation, e.g., epiblast, primitive streak or ectoderm was transient and
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it was difficult to obtain pure exact lineage outcomes due to the low induction efficiency. Although
we did not define the specific lineages in which miR-23a cluster members are up-regulated, we
demonstrated that miR-23a/b clusters were indispensible for cardiomyocyte differentiation of ESCs
and also required for mesoderm differentiation of ESCs in vivo. We consider that clarifying the
essential role of the microRNAs in specific lineage differentiation may be more important than
ascertaining their expression patterns in the specific lineages.
Summary
Overall, the authors have done a thorough and careful job in parsing the role of miR-27a and miR24 and showed convincingly their role in targeting pluripotency genes. Therefore the large volume
of data in this study should further our understanding of pluripotency, differentiation and
microRNAs by identifying novel differentiation-inducing miRNAs. However, I believe the authors
should clarify whether these miRNAs are endogenously required during mESC differentiation to
silence self-renewal and execute differentiation (see above), amongst other points listed above and
some minor comments below, and I think addressing these concerns could further improve their
already impressive manuscript.
Minor comments
1. The distinction between wild type or DGCR8 mESCs should be made in the Figures 1, 2, 4, 5 and
their legend to clarify what cell type was used in the experiment. For example, indicate in figures 2a,
c, d, and 1d whether wild type or DGCR8-/- mESCs was used. In figure 3a, indicate 'wild type' in
the Figure.
The cell type used in the experiment has been added to the figure legends. “Wild-type ESC” has
also been indicated in the figure 2F (previous figure 3a).
2. Page 2, line 27 - The phrase "pre-selected collection of 40 microRNAs in Dgcr-8-null ESCs" in
the abstract needs to be clarified by including a brief statement of the criteria used to select these 40
microRNAs.
The phrase has been changed to “40 microRNAs pre-selected by their expression patterns and
predicted targets in Dgcr8-null ESCs”.
3. Page 2, line 51 - "ESCs lacking of key enzymes" with "ESCs lacking key enzymes"
The phrase has been corrected.
4. Figure 7e - correct miR-241# to miR-24 #1.
1# and 2# have been changed to #1 and #2 in the revised manuscript.
5. Include the detail that regulation of targets by miRNA is direct.
The detailed methods and results concerning Ago2-IP had been included in the manuscript (line:
250-265 and line: 615-640).
6. Page 7, line 147 "microRNA mimic treated ESCs" to "microRNA mimic-treated ESCs".
The phrase has been corrected. The similar mistakes throughout the manuscript have been
corrected.
7. Mention whether the rank from Figure 2e is based on the microarray data or real-time PCR
validation.
The rank in Figure 2E was not based on microarray or real-time PCR data. It was based on their
ability to silence ESC self-renewal including the data from colony formation assay, AP staining, cell
cycle, Oct4 staining and gene expression pattern. These items had been included in figure 2E and
supplementary table S7.
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8. Include negative control for Figure 5g. - a gene that is not regulated by miR-24 and miR-27a.
According to the suggestion of the reviewer, we detected the presence of GAPDH mRNA that was
not regulated by miR-27a/24 in the RNAs isolated from Ago2-IP through RT-PCR. The results
showed that GAPDH was not bound by Ago2 complex in both scramble and microRNA mimictreated Dgcr8-/- ESCs. Figure5G has been placed at Figure 4F and 4G in the revised manuscript.
9. Figure legend of Figure 2c may be clarified and explained. For example, is this gene expression
profile after microRNA overexpression in DGCR8-/- mESCs?
Yes, it was the gene expression profile of miRNA mimic-transfected Dgcr8-/- ESCs. This has been
added in the figure legends.
10. For Fig. 4c,e it would be useful to annotate those subpanels in the figure itself as being
conducted in DGCR8-/- mESCs, given that wild-type mESCs are also discussed in the
accompanying text.
In the revised manuscript, figure 4C and 4E were combined and placed as figure 3H which was
indicative of being conducted in Dgcr8-/- mESCs.
All the major changes in the revised manuscript are marked as red characters.
Response to Referee #3:
The manuscript by Ma et al. presents interestesting findings of several miRNAs capable of
suppressing ESC self-renewal and/or inducing their differentiation, and the conclusions are
supported by several different approaches. Particular focus is given to the miRNAs of the miR23a/miR-27a/miR-24 cluster and knowledge about their role in ESC differentiation might benefit
future work in field of cellular reprogramming as shown by the authors. The findings are of general
interest and manuscript for most parts well written. However, more details should be provided for
certain technical aspects, such as the exact Dgcr8-/- cell line used, and throughout the manuscript
the figure legends should be more detailed. Also, more clarafication and analysis would be needed
especially on selection of the miR-23a/miR-27a/miR-24 cluster and on the experiments about the
transcriptional regulation of this cluster by c-Myc.
More detailed major comments are listed below:
1) Some bioinformatic analysis require more details in Materials&Methods or Figure Legends. For
example, from TargetScan predictions in Supplementary Table S3, which exact tool, which version,
and which settings were considered for selecting binding sites (seems all possible were included).
Also, which RNA structure prediction software was used for miR-23a/miR-27a/miR-24 cluster
structure in Fig3B? No reference/link is provided. What message do the authors want to convey with
this panel (3B)? Please explain the details of this (and most other) figure legends better.
We are sorry for the incomplete methods or figure legends. TargetScan Mouse 6.2
(http://www.targetscan.org/mmu_61/) was used to view the miRNA sites in the 3’UTR of these
pluripotency-associated transcription factors and all possible miRNA sites were included. The RNA
structure prediction software we used was “RNA structure”
(http://rna.urmc.rochester.edu/RNAstructureWeb/) and the link was supplied in the new version of
Methods. The previous figure 3B has been deleted in revised manuscript. In addition, most of the
figure legends have been supplemented based on the suggestions of the reviewer.
2) The Dgcr8-deficient ES cell line, which is a central model system of this manuscript, is first
mentioned at page 6. However, no reference to the origin of the cell line is provided. This should be
included in minimum in the Materials and Methods. Have the authors received the cell line from
somewhere or created it themselves? If latter, all details of cell line and the knock-out should be
provided.
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-­‐/-­‐
The Dgcr8 ESCs were kindly provided by Dr.Yangming Wang who constructed the cell line at University of California, San Francisco in 2007 (Wang Y, et al. DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-­‐renewal. Nat Genet. 2007; 39:380-­‐5). He is also a co-­‐author of the manuscript. The detailed information and reference have been supplemented in the new version of Methods. 3) Fig 2C: This is another example of a figure panel that lacks sufficient details. Equally the details
are missing from the corresponding Supplementary Table S6. What values does the color bar or the
numbers in the table represent (fold change, linear or log scale, relative to what)? At least in the
supplementary table, also statistical significance of these changes should be provided, if the data are
based on biological triplicates.
The numbers in the Supplementary Table S6 represent the relative expression of these genes in
microRNA mimic-treated Dgcr8-/- ESCs compared with the scramble control. This explanation has
been added to the table and the statistical significances are also provided in the revised manuscript.
The heat map represents the fold change of these genes’ expression in microRNA mimic-transfected
Dgcr8-/- ESCs compared with the scramble control.
4) Page 8, "Grading and Scoring of screening results": This chapter is written somewhat vaguely and
description of Fig 2E should be more detailed given its central role for the manuscript (including
references to previous figures).
To quantitatively analyze the comprehensive data from colony formation assay, AP staining, cell
cycle, gene expression pattern and Oct4 staining, we firstly scored on each item and then added
together. As shown in Supplementary table S7, score on colony formation assay was the logarithm
of the ratio of the colony number relative to scramble control, with base 2. Score on AP staining
was in the range of -3 to 3, with -3 being maximal loss of staining and 3 being maximal boost of
staining. Score on cell proliferation was the logarithm of the ratio of G1 fractions (%) relative to
scramble control, with base 1.1. Taking 1.1 as base was to make the grade in the range of -6 to 6.
Score on Oct4 staining was in the range of -3 to 3, with -3 being maximal loss of staining and 3
being maximal boost of staining. Score on Oct4 expression was the logarithm of Oct4 mRNA level
relative to scramble control, with base 2. Score on Sox2 or Nanog expression was the logarithm of
Sox2 or Nanog mRNA level relative to scramble control, with base 4. The total score, which was
sum of the above mentioned scores on each item, can comprehensively reflect the ESC self-renewal
state in the miRNA mimic-transfected ESCs. We ranked the miRNAs based on the total score.
According to the nice suggestion of the reviewer, we have placed the detailed explanation for the
grading and scoring in the new version of Methods and Materials.
5) Page 9: Logic for selection of miR-23a/miR-27a/miR-24 cluster for further studies should be
better clarified. Why miR-23b/miR-27b/miR-24 cluster was not studied further although it also
expresses miR-24 and miR-27b has the same seed as miR-27a (and therefore presumably shares
many of its targets)? Is miR-23b/miR-27b/miR-24 cluster expressed with similar or different
patterns and what is its relative contribution to the total expression levels in different tissues,
including ESCs?
We appreciate the reasonable suggestions from the reviewer and agree with that miR23b~27b~24 cluster may be physiologically functional compensative or redundant with miR23a~27a~24 cluster as the same sequence at the seed region although the effect of miR-27a on ESC
self-renewal was stronger than that of miR-27b in the initial screening in Dgcr8-/- ESCs. In the
revised manuscript, both of miR-23a~27a~24 cluster and miR-23b~27b~24 cluster were studied for
their roles in ESC differentiation. Excitingly, the additional data about the deficiency in
differentiation of miR-23~27~24 cluster knockout ESCs suggested that miR-23b~27b~24 cluster
may resemble the role of miR-23a~27a~24 cluster in regulating ESC self-renewal and
differentiation (see Fig6 and Fig7 in revised manuscript).
According to the suggestion from the reviewer, we also detected the expression of miR23b~27b~24-1 cluster, i.e. miR-23b and miR-27b during ESC differentiation as miR-24 were the
common products of the two cluster. The results showed that miR-23b and miR-27b were upregulated during EB formation and RA induced differentiation which was similar to the expression
profile of miR-23a cluster. We also tested the relative contribution of the two clusters in mESCs and
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found that the expression of miR-23a/27a was approximately equivalent to that of miR-23b/27b. In
addition, we did not include the expression profile of miR-23b cluster in the revised manuscript due
to the excessive data.
A. The expression profile of miR-23b during EB formation and RA induced mESCs differentiation.
B. The expression profile of miR-27b during EB formation and RA induced mESCs differentiation.
C. The relative expression of miR-23a/23b, miR-27a/27b in mESCs.
6) Fig 5C: what is the positive control used in the reporter gene assay? Figure should be consistent
with legend which should be consistent with details from materials and methods.
The reverse complementary sequence of miR-27a and miR-24 was inserted into the pMIR-reporter
vector to generate reporters that can detect mature miRNA expression and were performed as
positive controls (PC) in 293T cells. This has been added in the new version of Methods and the
explanation for PC has been supplemented in the figure legends. The previous Fig. 5C has been
placed as Fig. 4C in the revised manuscript.
7) Fig 6C: What are the genomic coordinates and sequence of the miR-23a/miR-27a/miR-24 cluster
promoter and transcription start site considered for the experiments? Is the use of this transcription
start site in ESCs supported by existing publically available epigenomic data such as H3K4me3 or
H3K36me3 enrichments (e.g ENCODE)? Are there alternative transcription sites? Is the binding of
c-Myc to this transcription start site supported by existing publically available ChIP-Seq data for cMyc binding in ESCs?
The promoter region and transcription start site of miR-23a cluster in human had been identified
[Lee Y, et al., 2004, EMBO J, 23: 4051-4060]. Due to the highly conservation of the transcription
start region of this cluster between human and mouse, we recognized -60bp upstream of mature
miR-23a sequence (Chr8:84208504) as transcription start site of mouse miR-23a cluster according
to the study of human miR-23a cluster. In addition, th editor thought this point was not strictly
required. So we did not check the epigenomic data such as H3K4me3 or H3K36me3 enrichments
and ChIP-Seq data for c-Myc binding in ESCs.
Reference:
Lee Y, Kim M, Han J, Yeom KH, Lee S, Baek SH, Kim VN (2004) MicroRNA genes are transcribed
by RNA polymerase II. EMBO J 23: 4051-4060.
8) Fig 6D: Please indicate the locus of the used negative control region.
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The previous Figure 6D has been placed at Figure 4K. The locus of negative control in Figure 4K
has been indicated in the new version of Methods.
Additional minor comments:
1) Please avoid using unspecifying abbreviations (like etc.) in the abstract.
The abbreviation “etc.” has been deleted from the abstract.
2) there should always be space between numbers and units. For example, "100 nM" instead of
"100nM".
Spaces have been added between numbers and units throughout the revised manuscript.
Final Note: We have revised the manuscript according to the suggestions from the reviewer. In
addition, based on the suggestions from the editor and the other two reviewers, we also make the
following changes and supply some additional data in the revised manuscript:
1. We detected several self-renewal and differentiation markers using immunofluorescence in
miRNA transfected-wild-type ESCs to better understand the roles of these miRNAs in wild-type
ESCs (Fig 3I).
2. Excitingly, by using newly-developed CRISPR/Cas9technologywe generated miR-23a-27a-24-2
and/or miR-23b-27b-24-1 cluster knockout mESCs, which is more suitable in studying the
endogenous and physiological role of the cluster in mESC differentiation substantially (Fig 6, 7 and
Supplementary Fig S7, 8).
3. We included negative control that was not regulated by miR-24 and miR-27a in Ago2-IP
experiments (Fig 4F, G).
All the major changes in the revised manuscript are marked as red characters.
Editorial Decision # ??
06 October 2014
Apologies for the rather slow response on your revised version. The reason for this being that one of
the original referees was not available for a second assessment. Given that the paper was formally
transferred from EMBOreports, I decided to involve a new referee as to make sure that the study
would uphold to our distinct expectations.
As you will see from both sets of comments, a few more amendments as to illuminate the actual
molecular mechanisms have been requested. I therefore encourage you to check for potential mycbinding sites across the miR-23-27-24 locus (see ref#1). Similarly, a mostly bioinformatics
assessment of relevant TF's to explain the differentiation regulation function of these these miRNAs
in the DKOs would enrich the value of the study (see ref#2s comments).
I look forward to receive your ultimately amended version in a rather timely manner.
REFEREE REPORTS:
Referee #1:
During revision the manuscript has been considerably further improved, in particular through the
individual and bi-allelic knock-outs of microRNA clusters in ES cells, and I would recommend
publication pending minor revisions based on comments below.
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1) While authors clearly show dependence of miR-23-27-24 cluster expression on c-Myc, it is very
likely to be mediated through multiple distal enhancers with binding sites for c-Myc across miR-2327-24 locus. To shed more light into this regulation and to strengthen the argument, inspection of
publicly available ChIP-Seq data for c-Myc binding in ES cells should be useful. These data are
readily accessible.
2) The genomic coordinates provided for the negative control region used in ChIP-PCR experiments
(chr8:82407127-84207294) spans a region of 1.8 Mb. There must be a typo in the coordinates.
Referee #2:
In this manuscript, Ma et al., performed a focused screen of 40 selected miRNAs to identify
miRNAs that that can inhibit self-renewal and promote the differentiation of miRNA deficient
(Dgcr8-/-) ESCs. By introducing synthetic miRNA mimics into Dgrc8-deficient ESCs, they
investigated the potential function of individual miRNAs to inhibit ESC colony formation, decrease
alkaline phosphatase activity, reduce cell proliferation, decrease expression of pluripotency
transcripts, and in contrast, upregulate expression of lineage specific markers. Among these
candidates, 7 miRNA (let-7c, miR-300-5p, 24-3p, 27a-3p, 124-3p. 27b-3p and 129-5p) also led to
decreased alkaline phosphatase activity when introduced to wild type V6.5 ESCs.
The authors followed up with more detailed studies on the role of miR-27a and miR-24 in ESCs.
These miRNAs are expressed from a cluster that also contains miR-23. Expression of these miRNAs
is induced during ESC differentiation and their introduction into Dgcr8-deficient ESCs, and to a
much lesser extent wild type ESCs, can induce ESC differentiation. The authors identified that these
miRNAs directly target pluripotency-associated transcription factors including Oct4 and Foxo1 as
well as two cell-signaling components in ESCs (gp130, and Smads) and suppress the pluripotency
program to initiate lineage differentiation. They used luciferase reporter assays with the relevant
3'UTRs to confirm the possible direct miRNA-mediated regulation. The direct binding of miR-27a
and miR-24 was confirmed by ribonucleoprotein-immunoprecipitation (RNP-IP) using anti-Ago2
antibody in Dgcr8-deficient ESCs.
Antagonizing miR-27a and miR-24 with antisense oligonucleotides could modestly (~2-fold)
enhance the reprogramming of mouse embryonic fibroblasts (MEFs) to induced pluripotent stem
cells (iPSCs). And the authors use a panel of assays to characterize the pluripotency of the iPSCs
generated. Finally, the authors used CRISPR/Cas9 technology to delete both the miR-23a, -27a, and
-24-2 and miR-23b miR-23b, -27b, and -24-1 loci from chromosomes 8 and 13, respectively, in
ESCs. Double knockout ESCs had severe deficiencies in differentiation toward mesodermal and
cardiac lineage. This was shown using a combination of morphological and gene expression
analyses as well as immunocytochemistry and histology.
Overall this is an interesting manuscript and the quality of the presented data is good and well
controlled. Although conceptually the approach (with the exception of the CRISP experiment) is not
especially original the authors provide some interesting new results. They furthermore perform
state-of-the-art genome editing to delete the two miR-23-24-27 loci to reveal a striking
differentiation phenotype. Therefore, I consider this manuscript suitable for publication in EMBO
Journal.
Specific comments to be addressed:
1. The miR-23-24-27 knockout phenotype is striking. The results convincingly show that these
miRNAs are involved in the lineage commitment to mesoderm. However, how this happens was not
shown in the manuscript. Indeed, the presented data only support negative regulation of pluripotency
genes by miRNAs. The authors need to explain how specifically differentiation to mesoderm is
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impaired in the double knockout cells. Is there any seed sequence in the 3' UTR of the
endoderm/ectoderm transcription factors? This is especially interesting because in the double KO
ESCs, expression of Mash1 and Nestin (ectoderm markers) are increased up to 30-fold at 8d EBs
(Figure 7A). A similar pattern is observed for expression of endodermal marker Foxa2 (around 50fold). Alternatively, these miRNAs might be involved in the suppression of cell signaling cascades
necessary for establishment of mesodermal lineage (Smads?).
2. In addition, it is not clear how many miRNA mimics were used for each individual miRNA? How
many knockout and double knockout cell lines were used in the experiments in the figures 6 and 7?
Authors need to clarify these issues.
Revision
28 October 2014
Referee #1:
During revision the manuscript has been considerably further improved, in particular through the
individual and bi-allelic knock-outs of microRNA clusters in ES cells, and I would recommend
publication pending minor revisions based on comments below.
1) While authors clearly show dependence of miR-23-27-24 cluster expression on c-Myc, it is very
likely to be mediated through multiple distal enhancers with binding sites for c-Myc across miR-2327-24 locus. To shed more light into this regulation and to strengthen the argument, inspection of
publicly available ChIP-Seq data for c-Myc binding in ES cells should be useful. These data are
readily accessible.
According to the helpful suggestion from the reviewer, we employed publicly available c-Myc ChIPSeq data in mESCs (Chen X et al, 2008), in which ChIP-qPCR validations were used to refine the
threshold to determine the specificity of binding site was greater than 98%. We analyzed 200kb
region across miR-23a~27a~24-2 cluster locus for c-Myc binding and found that c-Myc can bind to
-60kb, -24kb and 48kb sites around the cluster. In consideration of the high fidelity and potential
false negatives, we re-analyzed the c-Myc ChIP-Seq raw data and reserved all the uniquely mapping
tags but discarding signals of anti-GFP (NC) ChIP. The results indicate that there are also some cMyc binding sites nearby miR-23a~27a~24-2 cluster, which needs to be validated further. Taken
together, these results suggest that c-Myc may regulate the expression of miR-23a~27a~24-2 cluster
not only through the -15bp proximal site but also through the multiple binding sites (figure S5D).
The results have been added to the revised manuscript.
Supplementary Fig S5D The potential c-Myc binding sites in 200kb region across miR23a~27a~24-2 cluster in mESCs.
Reference
Chen X, Xu H, Yuan P, Fang F, Huss M, Vega VB, Wong E, Orlov YL, Zhang W, Jiang J, Loh YH,
Yeo HC, Yeo ZX, Narang V, Govindarajan KR, Leong B, Shahab A, Ruan Y, Bourque G, Sung WK,
Clarke ND, Wei CL, Ng HH (2008). Integration of external signaling pathways with the core
transcriptional network in embryonic stem cells. Cell 133:1106-1117.
2) The genomic coordinates provided for the negative control region used in ChIP-PCR experiments
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(chr8:82407127-84207294) spans a region of 1.8 Mb. There must be a typo in the coordinates.
We apologized for the typo. It has been corrected to chr8:84207127-84207294.
Final Note: We have revised the manuscript according to the suggestions from the reviewer. In
addition, according to the suggestions from the other reviewer, we discussed the potential
mechanism of miR-23/27/24 in regulating mesoderm lineage commitment through comprehensive
analysis of their targets (Supplementary Fig S9) and also summarized ESC clones with different
genotypes generated by CRISPR/Cas9 in Fig 6B.
All the major changes in the revised manuscript are marked as red characters.
Referee #2:
In this manuscript, Ma et al., performed a focused screen of 40 selected miRNAs to identify
miRNAs that that can inhibit self-renewal and promote the differentiation of miRNA deficient
(Dgcr8-/-) ESCs. By introducing synthetic miRNA mimics into Dgrc8-deficient ESCs, they
investigated the potential function of individual miRNAs to inhibit ESC colony formation, decrease
alkaline phosphatase activity, reduce cell proliferation, decrease expression of pluripotency
transcripts, and in contrast, upregulate expression of lineage specific markers. Among these
candidates, 7 miRNA (let-7c, miR-300-5p, 24-3p, 27a-3p, 124-3p. 27b-3p and 129-5p) also led to
decreased alkaline phosphatase activity when introduced to wild type V6.5 ESCs.
The authors followed up with more detailed studies on the role of miR-27a and miR-24 in ESCs.
These miRNAs are expressed from a cluster that also contains miR-23. Expression of these miRNAs
is induced during ESC differentiation and their introduction into Dgcr8-deficient ESCs, and to a
much lesser extent wild type ESCs, can induce ESC differentiation. The authors identified that these
miRNAs directly target pluripotency-associated transcription factors including Oct4 and Foxo1 as
well as two cell-signaling components in ESCs (gp130, and Smads) and suppress the pluripotency
program to initiate lineage differentiation. They used luciferase reporter assays with the relevant
3'UTRs to confirm the possible direct miRNA-mediated regulation. The direct binding of miR-27a
and miR-24 was confirmed by ribonucleoprotein-immunoprecipitation (RNP-IP) using anti-Ago2
antibody in Dgcr8-deficient ESCs.
Antagonizing miR-27a and miR-24 with antisense oligonucleotides could modestly (~2-fold)
enhance the reprogramming of mouse embryonic fibroblasts (MEFs) to induced pluripotent stem
cells (iPSCs). And the authors use a panel of assays to characterize the pluripotency of the iPSCs
generated. Finally, the authors used CRISPR/Cas9 technology to delete both the miR-23a, -27a, and
-24-2 and miR-23b miR-23b, -27b, and -24-1 loci from chromosomes 8 and 13, respectively, in
ESCs. Double knockout ESCs had severe deficiencies in differentiation toward mesodermal and
cardiac lineage. This was shown using a combination of morphological and gene expression
analyses as well as immunocytochemistry and histology.
Overall this is an interesting manuscript and the quality of the presented data is good and well
controlled. Although conceptually the approach (with the exception of the CRISP experiment) is not
especially original the authors provide some interesting new results. They furthermore perform
state-of-the-art genome editing to delete the two miR-23-24-27 loci to reveal a striking
differentiation phenotype. Therefore, I consider this manuscript suitable for publication in EMBO
Journal.
Specific comments to be addressed:
1. The miR-23-24-27 knockout phenotype is striking. The results convincingly show that these
miRNAs are involved in the lineage commitment to mesoderm. However, how this happens was not
shown in the manuscript. Indeed, the presented data only support negative regulation of pluripotency
genes by miRNAs. The authors need to explain how specifically differentiation to mesoderm is
impaired in the double knockout cells. Is there any seed sequence in the 3' UTR of the
endoderm/ectoderm transcription factors? This is especially interesting because in the double KO
ESCs, expression of Mash1 and Nestin (ectoderm markers) are increased up to 30-fold at 8d EBs
(Figure 7A). A similar pattern is observed for expression of endodermal marker Foxa2 (around 50-
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fold). Alternatively, these miRNAs might be involved in the suppression of cell signaling cascades
necessary for establishment of mesodermal lineage (Smads?).
We appreciated the helpful suggestions from the reviewer and discussed the potential mechanism of
miR-23/27/24 regulating mesoderm lineage commitment through comprehensive analysis of their
targets. We predicted all the potential targets of miR-23/27/24 through TargetScan and overlapped
them with FunGenES Database (http://biit.cs.ut.ee/fungenes/) (Schulz H et al, 2009), in which
transcriptome of mouse ESCs in sixty-seven experimental conditions (including differentiation to
three germ layers) were analyzed and organized in an interactive pattern. The database generated
clusters of transcripts that behave the same way under the entire spectrum of the sixty-seven
experimental conditions. As a result of overlapping the potential targets of miR-23/27/24 with
FunGenES Database, the predicted targets are greatly enriched in the clustered transcripts
differentially expressed in ESCs under the sixty-seven experimental conditions. The percentage of
miR-23/27/24 targets in all the mouse genes is about 2.58%, whereas the percentage gives rise to
12.79% when compared with the clusters generated by the Database. This suggests that the
potential targets of miR-23/27/24 are closely related to ESC state and differentiation. We also
compared the enrichment of the targets in the individual clusters and ranked the enriched clusters
with >30 transcripts. The results indicate that the potential targets are more enriched in cluster 14,
4 and 6 than the others (Supplementary Fig 9A, B). Among them, cluster 14 decreases obviously
during early mesoderm differentiation and increases a little during the further adipocyte
differentiation, while exhibits high expression during neuronal and pancreatic differentiation;
cluster 4 shows coincident decreased expression during early mesoderm differentiation and
subsequent adipocyte differentiation, whereas exhibits high expression under neuronal and
pancreatic differentiation conditions; cluster 6 behaves the similar way as cluster 4 except for
higher expression at day11 of adipocyte differentiation (Supplementary Fig 9C). Expression profile
of the enriched clusters indicate that miR-23/27/24 regulate mesoderm differentiation of ESCs
possibly through down-regulating the positive regulators of ectoderm or endoderm differentiation,
meanwhile down-regulating the negative regulators of mesoderm differentiation. This is consistent
with their critical roles in mesoderm lineage commitment. So miR-23/27/24 KO ESCs showed
increased expression of some ectoderm and endoderm markers. The detailed mechanism of their
regulatory roles in ESC mesoderm differentiation and cardiomyocyte differentiation will be studied
further. The discussion about this has been added to the revised manuscript.
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Supplementary Figure S9 Comparative analysis of predicted targets of miR-23/27/24 with
FunGenES Database. A, B. Distribution of the predicted targets of miR-23/27/24 among the
individual clusters with >30 transcripts, generated by FunGenES Database. C. The expression
profile of the clusters during ESC differentiation.
Reference
Schulz H, Kolde R, Adler P, Aksoy I, Anastassiadis K, Bader M, Billon N, Boeuf H, Bourillot PY, Buchholz F, Dani C, Doss MX, Forrester L, Gitton M, Henrique D, Hescheler J, Himmelbauer H, Hübner N, Karantzali E, Kretsovali A, Lubitz S, Pradier L, Rai M, Reimand J, Rolletschek A, Sachinidis A, Savatier P, Stewart F, Storm MP, Trouillas M, Vilo J, Welham MJ, Winkler J, Wobus AM, Hatzopoulos AK; Functional Genomics in Embryonic Stem Cells Consortium (2009) The FunGenES database: a genomics resource for mouse embryonic stem cell differentiation. PLoS One 4:e6804. 2. In addition, it is not clear how many miRNA mimics were used for each individual miRNA? How
many knockout and double knockout cell lines were used in the experiments in the figures 6 and 7?
Authors need to clarify these issues.
MicroRNA Mimics used in our experiments are ordered from Dharmacon (Thermofisher) and are
double-stranded RNA molecules with the same sequence with native miRNAs. One miRNA mimic
was used for each individual miRNA and each experiment was repeated at least three times. In
addition, we summarized ESC clones with different genotypes generated by CRISPR/Cas9 in Fig 6B.
One knockout and one double knockout cell line, total of 2 cell lines were used in the further
experiments in the figures 6 and 7. These have been indicated in the revised materials and results.
Final Note: We have revised the manuscript according to the suggestions from the reviewer. In
addition, we also employed publicly available c-Myc ChIP-Seq data in mESCs to analyze the
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multiple potential c-Myc binding sites across miR-23a~27a~24-2 cluster locus according to the
suggestions from the other reviewer (Supplementary Fig S5D).
All the major changes in the revised manuscript are marked as red characters.
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