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The EMBO Journal Peer Review Process File - EMBO-2013-86001
Manuscript EMBO-2013-86001
Ibf1 and Ibf2 are novel CP190-interacting proteins required
for insulator function.
Sergi Cuartero, Ujué Fresán, Oscar Reina, Evarist Planet, M Lluisa Espinás
Corresponding author: M Lluisa Espinás, IBMB-CSIC
Review timeline:
Submission date:
Editorial Decision:
Revision received:
Accepted:
17 June 2013
17 July 2013
14 November 2013
18 December 2013
Editor: Hartmut Vodermaier/Anke Sparmannn
Transaction Report:
(Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity,
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Editor:
1st Editorial Decision
17 July 2013
Thank you for submitting your research manuscript entitled " Ibf1 and Ibf2 are novel CP190interacting proteins required for insulator function" (EMBOJ-2013-86001) to our editorial office. It
has now been seen by three referees and their comments are provided below.
All reviewers appreciate your study and are in general supportive of publication in The EMBO
Journal. Nevertheless, they do raise a number of important concerns, and emphasize that a
significant revision of the manuscript will be required. In particular, your genetic analysis should be
extended according to the constructive suggestions of referee 2 and the effects of Ibf depletion on
genome-wide CP190 binding need the be further investigated.
Given the comments provided, I would like to invite you to submit a suitably revised manuscript to
The EMBO Journal that attends to the raised concerns in full. I should add that it is our policy to
allow only a single major round of revision and that it is therefore important to address the raised
concerns at this stage. Please do not hesitate to contact me should any particular argument require
further clarification.
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We generally allow three months as standard revision time. As a matter of policy, competing
manuscripts published during this period will not negatively impact on our assessment of the
conceptual advance presented by your study. However, we request that you contact the editor as
© European Molecular Biology Organization
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The EMBO Journal Peer Review Process File - EMBO-2013-86001
soon as possible upon publication of any related work, to discuss how to proceed. Should you
foresee a problem in meeting this three-month deadline, please let us know in advance and we may
be able to grant an extension.
Thank you for the opportunity to consider your work for publication. I look forward to your
revision.
_____
REFEREE REPORTS:
Referee #1:
The manuscript by Cuartero et al describes the characterization of two new insulator proteins in
Drosophila. They first isolate protein complexes containing a tagged form of the insulator proteins
CP190 and they carry out proteome analyses to identify to proteins they call Ibf1 and Ibf2. They go
on to show that these proteins are DNA-binding BED finger-containing factors and that they overlap
with a fraction of CP190 in chromosomes by IF as well as ChIP-seq. Mutations in the Ibf1/2 genes
affect insulator function as well as recruitment of CP190. Overall, the results are interesting and
nicely support the conclusions. A few minor concerns are described below:
1. The authors mention that the effect of RNAi of Ibf1/2 and CP190 on transcription was modest.
This could be due to residual amounts of Ibf1/2 or CP190 after dsRNA treatment. The authors
should include western analyses of treated cells to show the reduction in Ibf1/2 and CP190 levels.
2. If Ibf1/2 are only present at 20-25% of CP190 sites, perhaps only a subset of CP190-regulated
genes should be affected in KD Ibf1/2 cells. Why are the same genes affected by KD of both
proteins?
3. The description on page 8 of the statistical analysis of the RNAi data is confusing and difficult to
follow.
4. The chromosomes in Figure 6 are difficult to see. There is a lot of space between the various
panels. Each panel could probably be enlarged, and the space between panels decreased, while
maintaining the size of the figure the same
Referee #2 :
CP190 is a landmark of insulators in Drosophila and mammals. While in the vertebrate systems,
CP190 is always associated with sites bound by CTCF, the context is a bit more complex in flies
where CP190 can be associated with insulator proteins such the Su(Hw), BEAF-32 or GAGA factor
(in addition to CTCF). Furthermore, there are much more CP190 decorated sites in flies than sites
marked by Su(Hw), BEAF-32, GAGA factor and CTCF, suggesting the existence of additional
factors responsible for CP190 recruitment. Using CP190 associated protein complex purification
procedures and mass-spec, the authors of the present study describe the identification of 2 new
related proteins that they named Ibf1 and Ibf2 (Insulator-binding-factor). The authors performed an
a number of biochemical, genome-wide and genetic experiments to demonstrate that Ibf1 and Ibf2
are involved in insulation function along with CP190. Overall I find the work very interesting and
convincing that Ibf1 and Ibf2 are indeed factors involved in CP190 recruitment and insulation
function. Given the interest in insulator and genome organization, I believe that this kind of work is
suitable for the readership of EMBO.
Given my interest into the genetic analysis of boundary/insulator function in Drosophila, most of my
comments focus on the genetic analysis of Ibf1 and Ibf2. The transcription units of both genes are
encoded from the same locus with their polarity in opposite and diverging orientation. Their
transcriptionnal start site (TSS) are in very close proximity, being 470 nucleotides apart from each
other. The authors used 2 P-elements insertions within each of the 2 genes respectively to induce
mutations by imprecise excision. The deletion induced in Ibf1 is unfortunately not instructive
because it also affect the downstream gene VhaM8,9. For the case of Ibf2, the initial P-element is
inserted within the open-reading frame of the gene, giving rise to the equivalence of a knock out
allele. They isolated a white -/- derivative of this allele to perform the genetic interaction with the
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Fab-8 insulator assay. To do so, the authors recovered white minus flies that kept the transposon.
Not enough details are given in the procedure. Very often such white derivative can remove adjacent
DNA from one side of the P-element. It is therefore important that the authors state in the material
and methods that they have check the integrity of the insertion site on both side of the insert.
Without this information one is left with the possibility that a deletion may have occurred further
downstream towards CG16376 or upstream, towards Ibf1 The authors should have isolated precise
excision events to monitor for reversion. Such revertant chromosome can be then used to verify for
background effects. This is particularly important when one deals with very weak phenotypes such
as those reported below. In general the whole section dealing with the generation of mutant alleles
by mobilization of P-elements is not described in enough details. How many chromosomes were
screened? Given the proximity of the 2 TSSs, it is very unfortunate that the authors did not manage
to recover a lesion affecting both genes. I am not requesting the isolation of such an allele for
acceptance of the manuscript, but the authors should consider following my advices at the end of my
review.
The recovery of mutant alleles allows assaying for insulation function in vivo. As insulator the
authors used Fab-8 from the bithorax complex that was pulled out in their ChIP-seq experiment. In
these experiments the Fab-8 insulator is placed between the white enhancer and the miniwhite gene
and insulating strains are selected on the basis of their light eye color. Once again the authors do not
provide enough details about the procedure. How many transformants did they recovered and what
is the proportion of them that give an appropriate eye color for further screening. The authors use
then their Ibf1 and 2 mutant strains to test for a release of insulation as revealed by darkening of the
eye color. Weak darkening of the eye color of the Fab-8 reporter construct is observed in
heterozygotes Ibf1/+ and Ibf2/+. The weakness of the eye color change is acceptable because the
mutants are tested in heterozygous conditions. But to accept such weak genetic interaction, the
authors should document it with another independent Fab-8 P-element insert. They should also test
precise excision alleles of Ibf1 and Ibf2 to rule out background effect. But a much more convincing
experiment would be to show us the eye color of the Fab-8 P-element into the context of an
Ibf2GCV17 homozygous or even better in an Ibf2GSV17/ Df(3R)ED5339. The construction of such
stocks should straightforward.
Fab-8 is one of very few insulators that have been identified on the basis of a phenotype when
deleted. Fab-8 separates iab-7 from iab-8, the 2 segments-specific functions of the bithorax complex
responsible for the specification of the 7th and 8th abdominal segments (A7 and A8). When Fab-8 is
deleted iab-7 and iab-8 fuse together, giving rise to flies in which A7 and A8 specification is
affected. There is a mixture of gain-of and loss-of-function phenotypes that are visible in females.
Gain-of-function is monitored on the dorsal side of A7, which is transformed towards A8, giving
rise to females that lack a 7th tergite. The loss-of-function phenotype is visible on the ventral side
where A7 is transformed into A6. This transformation is monitored by the shape of the 7th sternite,
that looks like the sternite of A6. The authors report here a loss-of-function phenotype of A7 toward
A6 in 70% of the female homozygous for Ibf2. They do not observe any change on the dorsal side.
This is a very weak effect and one obviously worries about background effect. The authors should
mount cuticles, as the one displayed in our paper (Barges et al. 2000) Does the initial line
p[GSV6]GS16482 show the same frequency of transformation? Another control would be to
monitor p[GSV6]GS16482 precise excision chromosome. Normally the authors should have
recovered such chromosome when they ran the P-element though dysgenesis to recover white minus
derivatives. I am not against reporting such weak effect but I believe that the involvement in Fab-8
insulation would be much more convincing if the authors had a strong darkening of the white
enhancer-blocker reporter in a Ibf2 homozygote.
Additional comments
Antibodies against Ibf1 and Ibf2 were generated in this study and used to perform Chip-seq
experiments. I would like to see a full western with both antibodies showing whole length of the
slot.
I wonder why the retarded complex migrates slightly faster in the presence of competitor DNA in
the EMSA experiments shown in Fig 5D.
Co localization of Ibf1 and Ibf2 on salivary gland chromosomes would be easier to follow by
showing split chromosomes with the red and green channels.
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Do the authors have any idea why BEAF does not comes up in the mass-spec data?
It would be good to have a figure showing the genomic organization of Ibf1 and Ibf2 with the Pelements inserts used to generate mutants.
Some advice for further genetic analysis
With 2 TSSs in such close proximity (and a bit of luck), it should have been possible to isolate an
imprecise excision removing both TSSs. I am not asking the authors to perform a new screen for
acceptance of the paper, but they should tell us precisely how they proceeded. How many
chromosomes were screened? If the P-element used to create the double-strand break is in trans with
a deletion of the regions, [such as Df(3R)ED5339] the proportion of imprecise excision increases.
Did the authors used such scheme. Precise excision should also be isolated to verify for background
effects. The experiments shown in Figure 6 indicate the likelihood that the Ibf2 allele may well
correspond to a double Ibf1,Ibf2 mutant. But it is only an indication and the proof will only come
from the generation of a double KO chromosome . With 2 TSSs in such close proximity, a CRISP-R
directed mutagenesis seems quite straightforward to design and perform.
Geneva, July 9 2013, FranÁois Karch
Referee #3:
CP190 appears to be a key factor in insulator function in Drosophila, apparently mediating
interactions among other insulator DNA-binding proteins. Its own binding to chromatin seems to be
in some cases dependent on other insulator proteins and in other cases independent. In this work, the
authors have asked if CP190 associates with proteins that might explain its chromatin binding
behavior. Tagged CP190 protein expressed in cultured cells was affinity purified and analysed by
mass spectrometry. Two prominent components, among others, were found to be previously
undescribed proteins that the authors name Ibf1 and Ibf2. These are two small, related zinc finger
proteins encoded by adjacent genes. The paper shows that these proteins co-immunoprecipitate with
CP190 from nuclear extracts, are often found at the same sites as CP190 on polytene chromosomes,
co-localize with about one fourth of genomic CP190 sites in ChIP-seq experiments. It is very likely
therefore that Ibf1/2 provide one of the ways in which CP190 is targeted to specific chromosome
sites and are a significant part of the insulator circuitry in the Drosophila nucleus.
Overall, the paper is rather long and dwells excessively in discussions that seem to add little. I have
a number of specific comments.
- Ibf1 and 2 were identified by mass spectrometric analysis of affinity purified complexes. These
were produced by expressing a tagged CP190 from the metallothionein promoter. No evaluation is
given of the level of expression: is it comparable with endogenous levels? Overexpression can lead
to anomalous interactions.
- p. 7-8. The lengthy discussion of CP190 and Ibf2 co-regulation analysis seems less than useful,
given the level of noise, the large number of CP190 sites that do not bind Ibf2 and the lack of
agreement in sequence motifs associated with the binding sites.
- The results show that Ibf1 and 2 co-immunoprecipitate with one another, implying that they form a
heterodimer at least part of the time. They almost always bind together, rarely without CP190 (1529% of Ibf sites) and they show a preference for CP190 sites containing CTCF rather than CP190SU(HW) or CP190-BEAF sites. Found at only some of the CP190 standalone sites. Overall,
however, there are many CP190 binding sites, including standalone sites, that still require
explanation and suggest that there may be additional unknown DNA-binding proteins involved.
- Table 1 and Table S1. The mass spec results of two CP190 purifications are surprisingly different.
It is odd that in one experiment CTCF is associated with CP190 but no SU(HW) while in the other
there is SU(HW) but not CTCF. Why such discrepancies?
- The polytene experiments do not add anything to the more specific ChIP-seq results and could well
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be cut.
- p. 6, line 3. This sentence is unclear. Presumably it means that some sites bind Ibf2 distinctly more
than Ibf1, implying that Ibf2 may also bind alone. But then the next sentence seems to contradict
this by saying that Ibf1 and 2 form hetero-oligomers. Are the authors suggesting that they can form
oligomers containing more Ibf2 than Ibf1? There is not much evidence for that. The simplest
scenario would be that CP190 can bind either Ibf1 or 2 but CP190 forms oligomers through its BTB
domain, therefore usually bringing together both Ibfs.
- p. 6, bottom of first paragraph. Here it is stated that by CjIP-seq 25% of CP190 sites also bind Lbfs
but, according to Figure S3, Ibf1/2 account for little more than one tenth of the total CP190 biding
sites reported by modENCODE. Is there an explanation for the much greater number of CP190 sites
found by modENCODE?
- Since the effects of insulator proteins on expression are relatively minor in general, it would have
been far more useful to do a careful analysis of the effects of Ibf depletion on CP190 binding: what
CP190 sites are in fact dependent on Ibfs? This would dispense with the unconvincing discussion of
statistics on p. 8.
- No details are given about the construct used to test insulator activity in vivo, no references given
for the Fab-8 insulator sequence.
- Figure 5B, C. The conserved motif enriched in Ibf binding sites is fairly simple and somewhat
degenerate. Even so, it is not very visible in the CP190 binding sites and only half of the CP190 sites
that also bind Ibfs contain the Ibf binding motif. This suggests that it directs the binding of CP190 in
only a small fraction of CP190 binding sites. What does the asterisk in front of CP190 mean?
- Figure 5D. Ibf1 and Ibf2 are each able to bind in the absence of the other, implying that they do not
need to form heterodimers to bind too DNA. Is there any evidence that they interact, other than the
co-IP from nuclear extracts?
- Figure 6G. This is the key experiment to show that Ibfs are important for CP190 binding but only a
few sites are shown. Genome-wide CP190 ChIP in the Ibf2 mutant should have been done. The
results suggest that even CP190 sites that also bind Ibfs are only partly dependent on Ibfs. These and
other results clearly imply that we still do not know how CP190 binds.
1st Revision - authors' response
14 November 2013
Referee #1 (Remarks to the Author):
The manuscript by Cuartero et al describes the characterization of two new insulator proteins in
Drosophila. They first isolate protein complexes containing a tagged form of the insulator proteins
CP190 and they carry out proteome analyses to identify to proteins they call Ibf1 and Ibf2. They go
on to show that these proteins are DNA-binding BED finger-containing factors and that they
overlap with a fraction of CP190 in chromosomes by IF as well as ChIP-seq. Mutations in the Ibf1/2
genes affect insulator function as well as recruitment of CP190. Overall, the results are interesting
and nicely support the conclusions. A few minor concerns are described below:
1. The authors mention that the effect of RNAi of Ibf1/2 and CP190 on transcription was modest.
This could be due to residual amounts of Ibf1/2 or CP190 after dsRNA treatment. The authors
should include western analyses of treated cells to show the reduction in Ibf1/2 and CP190 levels.
We agree with the referee that this is an important point and in this new version of the manuscript
we have included western analyses of RNAi treated cells (Fig. S6). These analyses show a partial
decrease in protein levels (2-3 fold) that could account for the modest effect on transcription
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observed as the referee suggested. On the other hand, small changes in gene expression have already
been reported for CP190 and other insulator proteins knockdowns which are consistent with our
results.
2. If Ibf1/2 are only present at 20-25% of CP190 sites, perhaps only a subset of CP190-regulated
genes should be affected in KD Ibf1/2 cells. Why are the same genes affected by KD of both
proteins?
Several of the differentially expressed genes are coincident between the two treatments but there is a
significant number of genes that are only affected in the case of CP190 depletion and some genes
that are only affected in the Ibf2 knockdown. As shown in Figure 3A genes that change significantly
their expression levels (those outside the blue circles) are mainly located in the upper right and
lower left quadrants, meaning that they are genes that change in the same direction in both
depletions. However, it must be noted that several of those differentially expressed genes do not fall
close to the diagonal meaning that they are highly affected in one knockdown but not in the other.
We agree with the referee that this was not specified in our previous version and we have now
explained this in more detail.
3. The description on page 8 of the statistical analysis of the RNAi data is confusing and difficult to
follow.
We agree with the referee that this statistical analysis might be confusing. We have shortened and
summarized the expression results deciding not to include the statistical analyses since, as pointed
by both referee 1 and 3, it does not help to interpret the data. Besides, we think it does not add much
to the understanding of the contribution of Ibf to insulator function and gene regulation.
4. The chromosomes in Figure 6 are difficult to see. There is a lot of space between the various
panels. Each panel could probably be enlarged, and the space between panels decreased, while
maintaining the size of the figure the same
Done.
Referee #2 (Remarks to the Author):
CP190 is a landmark of insulators in Drosophila and mammals. While in the vertebrate systems,
CP190 is always associated with sites bound by CTCF, the context is a bit more complex in flies
where CP190 can be associated with insulator proteins such the Su(Hw), BEAF-32 or GAGA factor
(in addition to CTCF). Furthermore, there are much more CP190 decorated sites in flies than sites
marked by Su(Hw), BEAF-32, GAGA factor and CTCF, suggesting the existence of additional
factors responsible for CP190 recruitment. Using CP190 associated protein complex purification
procedures and mass-spec, the authors of the present study describe the identification of 2 new
related proteins that they named Ibf1 and Ibf2 (Insulator-binding-factor). The authors performed an
a number of biochemical, genome-wide and genetic experiments to demonstrate that Ibf1 and Ibf2
are involved in insulation function along with CP190. Overall I find the work very interesting and
convincing that Ibf1 and Ibf2 are
indeed factors involved in CP190 recruitment and insulation function. Given the interest in insulator
and genome organization, I believe that this kind of work is suitable for the readership of EMBO.
Given my interest into the genetic analysis of boundary/insulator function in Drosophila, most of my
comments focus on the genetic analysis of Ibf1 and Ibf2. The transcription units of both genes are
encoded from the same locus with their polarity in opposite and diverging orientation. Their
transcriptionnal start site (TSS) are in very close proximity, being 470 nucleotides apart from each
other. The authors used 2 P-elements insertions within each of the 2 genes respectively to induce
mutations by imprecise excision. The deletion induced in Ibf1 is unfortunately not instructive
because it also affect the downstream gene VhaM8,9. For the case of Ibf2, the initial P-element is
inserted within the open-reading frame of the gene, giving rise to the equivalence of a knock out
allele. They isolated a white -/- derivative of this allele to perform the genetic interaction with the
Fab-8 insulator assay. To do so, the authors recovered white minus flies that kept the transposon.
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Not enough details are given in the procedure. Very often such white derivative can remove
adjacent DNA from one side of the P-element. It is therefore important that the authors state in the
material and methods that they have check the integrity of the insertion site on both side of the
insert. Without this information one is left with the possibility that a deletion may have occurred
further downstream towards CG16376 or upstream, towards Ibf1. The authors should have isolated
precise excision events to monitor for reversion. Such revertant chromosome can be then used to
verify for background effects. This is particularly important when one deals with very weak
phenotypes such as those reported below. In general the whole section dealing with the generation
of mutant alleles by mobilization of P-elements is not described in enough details. How many
chromosomes were screened? Given the proximity of the 2 TSSs, it is very unfortunate that the
authors did not manage to
recover a lesion affecting both genes. I am not requesting the isolation of such an allele for
acceptance of the manuscript, but the authors should consider following my advices at the end of my
review.
After mobilization of the P-element we did not look for precise excision events since we thought
that mobilization of the P-element would generate excision footprints which, being located in the
coding sequence of the Ibf2 gene, might affect the integrity of the gene. Thus, we only looked for
white minus flies that either kept part of the transposon or delete enough DNA sequences to also
affect Ibf1 gene. However, since we obtained similar effects in enhancer-blocking assays with both
Ibf2GSV17 and Ibf1DRB1 lines, which come from two totally different backgrounds, we think that our
phenotypes are due to the absence of the Ibf1/Ibf2 complex and not to background effects. We
obtained 40 white minus lines, among them, none with a lesion affecting both genes and 13 lines
that kept parts of the transposon. For our genetic analyses we characterized two of them, Ibf2GSV17
and Ibf2GSV20, and we checked by PCR the integrity of the genomic sequences on both sides of the
P-element. We agree with the referee that not enough details were given about the procedure in the
previous version of the manuscript and this is now explained in detail in the material and methods
section of the manuscript.
We agree with the referee that it was very unfortunate not recover a lesion affecting both genes and
we will follow his advice to try to obtain it in the framework of our future work.
The recovery of mutant alleles allows assaying for insulation function in vivo. As insulator the
authors used Fab-8 from the bithorax complex that was pulled out in their ChIP-seq experiment. In
these experiments the Fab-8 insulator is placed between the white enhancer and the miniwhite gene
and insulating strains are selected on the basis of their light eye color. Once again the authors do
not provide enough details about the procedure. How many transformants did they recovered and
what is the proportion of them that give an appropriate eye color for further screening. The authors
use then their Ibf1 and 2 mutant strains to test for a release of insulation as revealed by darkening
of the eye color. Weak darkening of the eye color of the Fab-8 reporter construct is observed in
heterozygotes Ibf1/+ and Ibf2/+. The weakness of the eye color change is acceptable because the
mutants are tested in heterozygous conditions. But to accept such weak genetic interaction, the
authors
should document it with another independent Fab-8 P-element insert. They should also test precise
excision alleles of Ibf1 and Ibf2 to rule out background effect. But a much more convincing
experiment would be to show us the eye color of the Fab-8 P-element into the context of an
Ibf2GCV17 homozygous or even better in an Ibf2GSV17/ Df(3R)ED5339. The construction of such
stocks should straightforward.
In this revised version of the manuscript we have included enhancer-blocking assays with another
independent Fab-8 transgenic line and another Ibf2GSV20 mutant (Fig. S7) and we provide
information in the material and methods section about the Fab-8 insulator transgenic lines we
obtained. Although we have not been able to perform enhancer-blocking assays in the context of an
Ibf2GSV17/ Df(3R)ED5339 since the stock of the deficiency we had carries an associated wmw gene,
we have performed these assays in Ibf2GSV17 homozygous conditions as the referee suggested. In
these experiments we use the F8109.6 line since both the Fab-8 transgene in line F8134.1 and the
Ibf2 gene are in chromosome 3. In these assays a significant increase in eye color was observed in
the homozygous condition since we obtained flies with red eyes. These experiments are now shown
in Figure 4C.
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Fab-8 is one of very few insulators that have been identified on the basis of a phenotype when
deleted. Fab-8 separates iab-7 from iab-8, the 2 segments-specific functions of the bithorax complex
responsible for the specification of the 7th and 8th abdominal segments (A7 and A8). When Fab-8 is
deleted iab-7 and iab-8 fuse together, giving rise to flies in which A7 and A8 specification is
affected. There is a mixture of gain-of and loss-of-function phenotypes that are visible in females.
Gain-of-function is monitored on the dorsal side of A7, which is transformed towards A8, giving rise
to females that lack a 7th tergite. The loss-of-function phenotype is visible on the ventral side where
A7 is transformed into A6. This transformation is monitored by the shape of the 7th sternite, that
looks like the sternite of A6. The authors report here a loss-of-function phenotype of A7 toward A6
in 70% of the female homozygous for Ibf2. They do not observe any change on the dorsal
side. This is a very weak effect and one obviously worries about background effect. The authors
should mount cuticles, as the one displayed in our paper (Barges et al. 2000) Does the initial line
p[GSV6]GS16482 show the same frequency of transformation? Another control would be to monitor
p[GSV6]GS16482 precise excision chromosome. Normally the authors should have recovered such
chromosome when they ran the P-element though dysgenesis to recover white minus derivatives. I
am not against reporting such weak effect but I believe that the involvement in Fab-8 insulation
would be much more convincing if the authors had a strong darkening of the white enhancerblocker reporter in a Ibf2 homozygote.
To show the loss-of-function phenotype of A7 toward A6 we have now mounted cuticles as the
referee suggested. We have also analyzed the frequency of transformation in the initial line and,
since we have not been able to perform analyses in precise excisions for the reason we explained
above, we have also checked the frequency of transformation in Ibf2GSV17/ Df(3R)ED5339
transheterozygotes and we have included these data in the revised version of the manuscript (page
11).
Additional comments
Antibodies against Ibf1 and Ibf2 were generated in this study and used to perform Chip-seq
experiments. I would like to see a full western with both antibodies showing whole length of the slot.
We agree with the referee that specificity of the antibodies is an important point and we have now
included western-blot analyses in Figure 1S to show specificity of aCP190, aIbf1 and aIbf2
antibodies.
I wonder why the retarded complex migrates slightly faster in the presence of competitor DNA in the
EMSA experiments shown in Fig 5D.
The analysis of our data suggests that the slight difference in migration is due, most likely, to
technical reasons.
Co localization of Ibf1 and Ibf2 on salivary gland chromosomes would be easier to follow by
showing split chromosomes with the red and green channels.
As pointed out by referee 3, the immunolocalization assays in polytene chromosomes presented in
Figure 1 do not add anything to the more specific ChIP-seq results and we have decided to eliminate
these data.
Do the authors have any idea why BEAF does not comes up in the mass-spec data?
To our knowledge there is no evidence in the literature of a BEAF-CP190 physical interaction. The
fact that nobody, including us, has been able to show a CP190-BEAF coimmunoprecipitation could
reflect either a technical unresolved issue or could be reflecting that CP190 and BEAF do not need
to interact to colocalize in the genome and to perform their chromatin functions.
It would be good to have a figure showing the genomic organization of Ibf1 and Ibf2 with the Pelements inserts used to generate mutants.
We have now presented this data in Figure S8.
Some advice for further genetic analysis
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With 2 TSSs in such close proximity (and a bit of luck), it should have been possible to isolate an
imprecise excision removing both TSSs. I am not asking the authors to perform a new screen for
acceptance of the paper, but they should tell us precisely how they proceeded. How many
chromosomes were screened? If the P-element used to create the double-strand break is in trans
with a deletion of the regions, [such as Df(3R)ED5339] the proportion of imprecise excision
increases. Did the authors used such scheme. Precise excision should also be isolated to verify for
background effects. The experiments shown in Figure 6 indicate the likelihood that the Ibf2 allele
may well correspond to a double Ibf1,Ibf2 mutant. But it is only an indication and the proof will
only come from the generation of a double KO chromosome . With 2 TSSs in such close proximity, a
CRISP-R directed mutagenesis seems quite straightforward to design and perform.
Geneva, July 9 2013, François Karch
We agree with the referee that the generation of a double knockout chromosome will provide
valuable data. We are thankful to the referee for his advice and although due to time constraints we
have not been able to obtain it yet, we will follow his suggestions for further investigations.
Referee #3 (Remarks to the Author):
CP190 appears to be a key factor in insulator function in Drosophila, apparently mediating
interactions among other insulator DNA-binding proteins. Its own binding to chromatin seems to be
in some cases dependent on other insulator proteins and in other cases independent. In this work,
the authors have asked if CP190 associates with proteins that might explain its chromatin binding
behavior. Tagged CP190 protein expressed in cultured cells was affinity purified and analysed by
mass spectrometry. Two prominent components, among others, were found to be previously
undescribed proteins that the authors name Ibf1 and Ibf2. These are two small, related zinc finger
proteins encoded by adjacent genes. The paper shows that these proteins co-immunoprecipitate with
CP190 from nuclear extracts, are often found at the same sites as CP190 on polytene chromosomes,
co-localize with about one fourth of genomic CP190 sites in ChIP-seq experiments. It is very likely
therefore that Ibf1/2 provide
one of the ways in which CP190 is targeted to specific chromosome sites and are a significant part
of the insulator circuitry in the Drosophila nucleus.
Overall, the paper is rather long and dwells excessively in discussions that seem to add little. I have
a number of specific comments.
- Ibf1 and 2 were identified by mass spectrometric analysis of affinity purified complexes. These
were produced by expressing a tagged CP190 from the metallothionein promoter. No evaluation is
given of the level of expression: is it comparable with endogenous levels? Overexpression can lead
to anomalous interactions.
At the time when the tagged expression of CP190 was done, we still hadn't generated specific
antibody to endogenous CP190, so the levels of expression were monitored by Western Blot using a
TAP antibody and choosing a moderate level of induction of the tagged protein. However, we
subsequently verified each interaction by co-IP using the specific CP190 antibody once we had
generated it. This is shown in the manuscript for CP190 interaction with Ibf1 and Ibf2 in Figure 1B.
- p. 7-8. The lengthy discussion of CP190 and Ibf2 co-regulation analysis seems less than useful,
given the level of noise, the large number of CP190 sites that do not bind Ibf2 and the lack of
agreement in sequence motifs associated with the binding sites.
As indicate above in the response to referee 1, we have shortened and summarized the expression
results deciding not to include the statistical analysis since, as pointed out by both referees, it does
not seem to help to interpret the data. Besides, we think it does not add much to the understanding of
the contribution of Ibf to insulator function and gene regulation.
- The results show that Ibf1 and 2 co-immunoprecipitate with one another, implying that they form a
heterodimer at least part of the time. They almost always bind together, rarely without CP190 (1529% of Ibf sites) and they show a preference for CP190 sites containing CTCF rather than CP190-
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SU(HW) or CP190-BEAF sites. Found at only some of the CP190 standalone sites. Overall,
however, there are many CP190 binding sites, including standalone sites, that still require
explanation and suggest that there may be additional unknown DNA-binding proteins involved.
We completely agree with the referee and we have now discussed this point on the revised version
of the manuscript:
“Nevertheless, there are still several CP190-binding sites that neither colocalize with previously
known insulator proteins nor with Ibf1 and Ibf2 suggesting that there may be additional unknown
DNA-binding proteins involved in insulator function in Drosophila.”
- Table 1 and Table S1. The mass spec results of two CP190 purifications are surprisingly different.
It is odd that in one experiment CTCF is associated with CP190 but no SU(HW) while in the other
there is SU(HW) but not CTCF. Why such discrepancies?
In the precipitation of CP190-TAP we applied stringent conditions to increase specifity, the
disadvantage being that some interactions can be affected or appear weakly. Thus, we think that
discrepancies are likely to be due to technical variability.
- The polytene experiments do not add anything to the more specific ChIP-seq results and could well
be cut.
We agree with the referee and we have decided to eliminate these data.
- p. 6, line 3. This sentence is unclear. Presumably it means that some sites bind Ibf2 distinctly more
than Ibf1, implying that Ibf2 may also bind alone. But then the next sentence seems to contradict this
by saying that Ibf1 and 2 form hetero-oligomers. Are the authors suggesting that they can form
oligomers containing more Ibf2 than Ibf1? There is not much evidence for that. The simplest
scenario would be that CP190 can bind either Ibf1 or 2 but CP190 forms oligomers through its BTB
domain, therefore usually bringing together both Ibfs.
We agree with the referee that this point was confusing in the previous version of the manuscript.
We think that the higher signals observed in the Ibf2 ChIP-seq analysis are neither due to binding of
Ibf2 alone nor to hetero-oligomers containing more Ibf2 than Ibf. They are likely to be due to
technical differences in the immunoprecipitation efficiency. We have modified the text to clarify
this point.
- p. 6, bottom of first paragraph. Here it is stated that by CjIP-seq 25% of CP190 sites also bind
Lbfs but, according to Figure S3, Ibf1/2 account for little more than one tenth of the total CP190
biding sites reported by modENCODE. Is there an explanation for the much greater number of
CP190 sites found by modENCODE?
We have applied a high level of stringency in the peak calling (only peaks above 100 reads above
the input were called) because our aim was to focus only on high confidence peaks to carry on our
analyses. As we show in this revised version of the manuscript in Figure S3, if we reduce this
threshold the number of peaks increases being closer to those found by ModEncode.
- Since the effects of insulator proteins on expression are relatively minor in general, it would have
been far more useful to do a careful analysis of the effects of Ibf depletion on CP190 binding: what
CP190 sites are in fact dependent on Ibfs? This would dispense with the unconvincing discussion of
statistics on p. 8.
We agree with the referee that this is an important point. Therefore, we have performed new ChIPseq analyses in wild-type and Ibf2GSV17 mutant larvae using aCP190 antibodies. These assays show
that CP190 is preferentially associated to Ibf sites in wild-type larvae confirming our previous
results. Moreover, comparison with CTCF, Su(Hw) and BEAF binding sites reveal that CP190 sites
that are lost in the absence of Ibf2 are preferentially CTCF sites while CP190 sites that are
maintained in the Ibf2 mutant condition preferentially colocalize with Su(Hw) sites. This is in
agreement with our results pointing to a special participation of Ibf1/Ibf2 complex in CP190
insulators that also contain CTCF. We have introduced these new data in the revised version of the
manuscript in Figure 6G, 6H and S10B.
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- No details are given about the construct used to test insulator activity in vivo, no references given
for the Fab-8 insulator sequence.
We have introduced references and details in the material and methods section about the Fab-8
insulator sequence and the transgenic lines used.
- Figure 5B, C. The conserved motif enriched in Ibf binding sites is fairly simple and somewhat
degenerate. Even so, it is not very visible in the CP190 binding sites and only half of the CP190 sites
that also bind Ibfs contain the Ibf binding motif. This suggests that it directs the binding of CP190 in
only a small fraction of CP190 binding sites. What does the asterisk in front of CP190 mean?
The asterisk means that the motif has been reported to be related to CP190 binding either directly or
by means of an unknown protein. We have now indicated this in the legend of Figure 5.
- Figure 5D. Ibf1 and Ibf2 are each able to bind in the absence of the other, implying that they do
not need to form heterodimers to bind too DNA. Is there any evidence that they interact, other than
the co-IP from nuclear extracts?
Ibf1 is able to bind to DNA in the absence of Ibf2 in vitro but, as we showed in the manuscript, it
needs the other to bind to chromatin in vivo. This is not due to a transcriptional effect because
mRNA levels remain unchanged whereas protein stabilization seems to be affected. These data and
the co-IP from nuclear extracts are in our opinion the best evidences that they interact in vivo.
- Figure 6G. This is the key experiment to show that Ibfs are important for CP190 binding but only a
few sites are shown. Genome-wide CP190 ChIP in the Ibf2 mutant should have been done. The
results suggest that even CP190 sites that also bind Ibfs are only partly dependent on Ibfs. These
and other results clearly imply that we still do not know how CP190 binds.
As we indicate above we have performed new ChIP-seq analyses in wild-type and Ibf2GSV17 mutant
larvae. These new analyses show that CP190 is preferentially associated to Ibf sites in wild-type
larvae confirming our previous results. These analyses also show that CP190 sites that are lost in the
absence of Ibf2 are preferentially CTCF sites while CP190 sites that are maintained in the Ibf2
mutant condition preferentially colocalize with Su(Hw) sites, indicating the complexity in the
mechanistic interdependences between insulator proteins. Altogether our studies reveal a novel
pathway of CP190 recruitment to specific chromosome sites although we agree with the referee that
further studies will be needed to completely decipher how CP190 binds.
Acceptance letter
18 December 2013
Thank you again for submitting your final revised manuscript and associated material for our
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-----------------------------------------------Referee #1:
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The manuscript represents an important contribution to the field of insulators in Drosophila. The
findings should eventually lead to a better understanding of how insulators affect gene expression.
The authors have addressed all the issues raised by the reviewers by clarifying or adding new
information. New experimental results address all the original concerns.
Referee #2:
The authors have provided satisfactory answers to my questions and concerns.
Referee #3:
The authors have respoded well to the reviewers' comments. I think the paper is much improved and
presents novel results of importance in the field of Drosophila insulators.
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