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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, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.) 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. When preparing your letter of response to the referees' comments, please bear in mind that this will form part of the Review Process File, and will therefore be available online to the community. For more details on our Transparent Editorial Process, please visit our website: http://www.nature.com/emboj/about/process.html 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 1 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 © European Molecular Biology Organization 2 The EMBO Journal Peer Review Process File - EMBO-2013-86001 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. © European Molecular Biology Organization 3 The EMBO Journal Peer Review Process File - EMBO-2013-86001 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 © European Molecular Biology Organization 4 The EMBO Journal Peer Review Process File - EMBO-2013-86001 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 © European Molecular Biology Organization 5 The EMBO Journal Peer Review Process File - EMBO-2013-86001 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. © European Molecular Biology Organization 6 The EMBO Journal Peer Review Process File - EMBO-2013-86001 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. © European Molecular Biology Organization 7 The EMBO Journal Peer Review Process File - EMBO-2013-86001 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 © European Molecular Biology Organization 8 The EMBO Journal Peer Review Process File - EMBO-2013-86001 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- © European Molecular Biology Organization 9 The EMBO Journal Peer Review Process File - EMBO-2013-86001 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. © European Molecular Biology Organization 10 The EMBO Journal Peer Review Process File - EMBO-2013-86001 - 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 consideration. In light of the positive re-review and all revisions and clarifications,l I am pleased to inform you that we have now accepted the study for publication in The EMBO Journal. Further information regarding the production and publication procedures can be found below. In particular, please make sure to send us the completed and signed license forms as early as possible, since we require them to be able to initiate the production process. Thank you again for this contribution to The EMBO Journal and congratulations on a successful publication! Please consider us again in the future for your most exciting work. -----------------------------------------------Referee #1: © European Molecular Biology Organization 11 The EMBO Journal Peer Review Process File - EMBO-2013-86001 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. © European Molecular Biology Organization 12