The Sae2/CtIP protein is required for efficient processing of DNA double-strand breaks that initiate homologous recombination in eukaryotic cells. Sae2/CtIP is also important for survival of single-stranded Top1-induced lesions and CtIP is known to associate directly with transcription-associated complexes in mammalian cells. Here we investigate the role of Sae2/CtIP at single-strand lesions in budding yeast and in human cells and find that depletion of Sae2/CtIP promotes the accumulation of stalled RNA polymerase and RNA-DNA hybrids at sites of highly expressed genes. Overexpression of the RNA-DNA helicase Senataxin suppresses DNA damage sensitivity and R-loop accumulation in Sae2/CtIP-deficient cells, and a catalytic mutant of CtIP fails to complement this sensitivity, indicating a role for CtIP nuclease activity in the repair process. Based on this evidence, we propose that R-loop processing by 5’ flap endonucleases is a necessary step in the stabilization and removal of nascent R-loop initiating structures in eukaryotic cells. Double-strand breaks in DNA are known to be lethal lesions in eukaryotic cells, and can be an important source of genomic instability during oncogenic transformation because of the possibility of misrepair, translocations, and rearrangements that initiate from these lesions . The repThe budding yeast enzyme Sae2 and its mammalian ortholog CtIP are important for DNA end processing in eukaryotes and has been shown to act in several ways to facilitate the removal of the 5ʹ strand at DNA double-strand breaks . PhosphoIn addition to the activities of Sae2/CtIP that promote MRX(N) functions, the protein has also been shown to possess intrinsic nuclease activity that is important for the processing of breaks, particularly those formed in the context of protein lesions, radiation-induced DNA damage, or camptothecin (CPT) damage during S phase . NucleasEukaryotic cells lacking Sae2 or CtIP were also observed years ago to be hypersensitive to topoisomerase one (Top1) poisons such as CPT . Top1 boIn recent years, it has become clear that transcription can play a major role in promoting genomic instability by forming stalled transcription complexes and RNA-DNA hybrids in the genome . Stable The Sen1 protein in budding yeast has been shown to regulate many aspects of RNA biology, including termination of RNA polymerase II transcription, 3′ end processing of mRNA, and dissociation of RNA-DNA hybrids . The humIn this work we sought to understand the mechanistic basis of the hypersensitivity of Sae2/CtIP-deficient cells to Top1 poisons and other forms of DNA damage, finding unexpectedly that the sensitivity of these cells can be partially suppressed by overexpression of Sen1/Senataxin or RNaseH. Genetic evidence in yeast and human cells shows that Sae2/CtIP-deficient cells require Senataxin and RNaseH enzymes for survival of genotoxic agents and that these cells exhibit high levels of RNA polymerase stalling and R-loop formation at sites of active transcription, consistent with a failure to recognize or process RNA-DNA hybrids. Based on this evidence we propose that Sae2/CtIP has an important role in processing R-loops that promotes the action of RNA-DNA helicases and ultimately cell survival after DNA damage.sae2Δ phenotype in yeast, we overexpressed several different RNA Pol II-associated factors in the mutant strain. We found that overexpression of the termination factor Sen1 markedly improved survival of the sae2Δ strain to genotoxic agents  background. A complete deletion of SEN1 is lethal . This analysis revealed small peaks of Sae2 enrichment, primarily in the S phase +CPT sample   overlap with the sites of RNA-DNA hybrids measured in wild-type yeast cells in a previous study (To determine if the relationship between  (MACS2)  after re (MACS2) . In cont (MACS2)  in compa (MACS2) . The enrus study .sae2Δ cells, possibly related to an accumulation of R-loops. To test this hypothesis we used the S9.6 antibody to detect RNA-DNA hybrids    to monitA damage . In contstalling . ImportaPDC1 or NRD1 genes, both known to be dependent on Sen1  of human Senataxin, the ortholog of yeast Sen1 that also acts to remove RNA-DNA hybrids and promotes transcription termination  that acts as a sensor for R-loops in the genome , a RNA polymerase II inhibitor, and found that inhibition of transcription rescues the R-loop accumulation phenotype caused by CtIP deficiency . As overD10R-E48R-mCherry protein in the absence of both CtIP and XPG.The XPG protein, a component of nucleotide excision repair , has beeD10R-E48R-mCherry sensor but in this case monitored its accumulation in undamaged cells by fluorescence activated cell sorting (FACS), using a modified technique reported previously with a fragment of RNaseH fused to GFP , a transcriptional co-regulator , and alsTo investigate the role of CtIP in R-loop accumulation at specific loci, we first generated an inducible genomic cassette containing a mouse class switch region (Sγ3) that has previously been shown to accumulate R-loops . We perfWe then examined several endogenous loci that have previously been reported to be prone to R-loop formation . The β-aSenataxin has been shown to localize at sites of transcription-replication conflicts and to travel with replication forks .Here we Our results suggest that the CtIP and XPG nucleases help to either prevent R-loop formation in the genome or to resolve R-loops once they are formed. Since CtIP and XPG are both specific for 5ʹ flaps, we considered a model in which CtIP and XPG process 5ʹ flaps present in an R-loop structure . We hypoOne prediction of this model is that CtIP and XPG-depleted cells would exhibit fewer single-strand DNA breaks compared to normal cells, even though they exhibit higher levels of R-loops. To test this idea, we created DNA damage with CPT and measured single-strand DNA breaks with an alkaline comet assay. We found that exposure to CPT generated a marked increase in DNA breaks, and that transcription promotes these breaks, as pretreatment with DRB reduced the levels of breaks by ~75% in wild-type cells . CtIP deNext, we assessed the levels of single-strand DNA breaks in cells depleted for XPG, or for XPG and CtIP concurrently. In cells depleted for XPG there are fewer breaks after CPT treatment, and in cells depleted for both proteins, we observed no increase in breaks at all with CPT exposure . This reTo further validate this result at a specific locus in the genome, we returned to the RPL13A gene, where we observed high levels of R-loops in cells lacking CtIP using DRIP-qPCR . Here weIn this study we demonstrate a functional relationship between the Sae2/CtIP enzyme and transcription-related DNA damage in eukaryotic cells, showing that the damage sensitivity of cells lacking Sae2/CtIP can be rescued by either Sen1/Senataxin or RNaseH overexpression. We further provide evidence for accumulation of stalled RNA polymerase complexes and RNA-DNA hybrids in cells deficient in Sae2/CtIP, with these lesions located at sites of highly expressed genes. These results suggest that Sae2/CtIP is not only a double-strand break resection factor, but also functions at transcription-associated lesions in eukaryotic cells.S. cerevisiae, the temperature-sensitive sen1-1 mutant exhibits synthetic lethality in combination with DNA repair mutants mre11, rad50, sgs1, and rad52, indicating a requirement for homologous recombination in the absence of SEN1 function . The sen1-1 strain and corresponding wild-type strain were gifts from Nicholas Proudfoot. The HTB-tagged Rpb2 and Sen1 strains were gifts from Jeff Corden. The rnh1 rnh201 mutant strain was a gift from David Tollervey. A high-copy vector containing the wild-type SAE2 gene with a 2xFLAG tag in pRS425 (61)‘FLAG-SAE2/2μ’ was a gift from John Petrini, and was used for the SAE2 ChIP experiment.For yeast strains see d pRS425  by fusio1), while the other half were washed and resuspended in fresh media (S). Previous studies have shown that all the cells enter S phase 30 min after the removal of alpha factor . After another 15 min CPT or DMSO was added to the media at indicated concentration.Cells were grown to exponential phase (OD 0.5–0.6) and synchronized using alpha-factor for 3 hr at 30°C. Half of the cells were kept in alpha factor (Ga factor . Therefo600 = 0.5–0.6. The cells were centrifuged and resuspended in 50 ml fresh media (concentrated 40-fold). Yeast mating pheromone alpha-factor was added to final concentration of 10 μM and cells were synchronized for 4 hr at 30°C. Half of the cells were kept in alpha factor (G1), while the other half were washed and resuspended in fresh media (S). After the wash, both G1 and S phase cells were further divided into two and treated with 100 μM camptothecin or DMSO for 50 min. At the end of the drug treatment cells were crosslinked by addition of formaldehyde  at RT for 25 min, followed by glycine  at RT for 5–10 min. The cells were harvested, washed with water and flash frozen until further processing. Thus 2L of starting culture was divided into four samples per strain with approximately 500 OD cells per sample.Sae2: Yeast cells containing a FLAG-Sae2 expression cassette on a high copy number 2μ plasmid were grown to exponential phase in 2 L of appropriate minimal media to ODEach cell pellet was resuspended in 400 μl lysis buffer  and lysed using a bead beater (3 cycles of 45 s) in the presence of 400 μl 0.5 mm zirconia beads. The beads were washed with 2 × 500 μl of lysis buffer to collect a total of 1.5 ml cell extract. Sonication was performed with a Branson Digital Sonifier . The cells were kept on ice for 5 min in between each cycle. The extract was cleared with high-speed centrifugation at 4°C for 30 min. A small sample was treated with proteinase K and RNaseA and separated by agarose gel to check for sonication efficiency (DNA size should be between 100–250 bp). The lysate was diluted to 10 ml with the lysis buffer. A 500 μl aliquot of the lysate was saved as the ‘Input’ sample. 50 μg Sigma FLAG-M2 antibody and 300 μl Pierce Protein A/G magnetic beads (prewashed with lysis buffer) were added to the cleared lysate. The cells were rotated at 4°C overnight. The following day, the beads were separated and washed 3 times each with wash-buffer 1 , wash buffer 2 , wash buffer 3  and wash buffer 4 . 500 μl elution buffer (wash buffer 4 + 3X FLAG peptide) was added to the beads and kept shaking at 4°C for 2 hr. The beads were eluted with 100 μl elution buffer (wash buffer 4 + 2% SDS) and incubated at 65°C for 30 min. A second elution was done with 100 μl elution buffer similarly and combined for 150 μl total elution. The final elution and the Input sample  were reverse cross-linked at 65°C overnight. Next day, all samples were diluted to SDS <0.5% and treated with 20 μg RNase A at 37°C 2 hr followed by 40 μg of proteinase K at 37°C 2 hr. The DNA was purified by phenol chloroform extractions followed by ethanol precipitation and stored at −20°C until further processing.The DNA samples were gel purified to obtain a size range of 50–200 bp. The sequencing libraries were prepared for each sample using NEBNext DNA Library prep kit for Illumina (E6040S) and were sequenced and analyzed at the UT Austin GSAF. The bam files and bed files were visualized using Integrated Genome Viewer (iGV) and compared with saccer3 reference genome. Primary data is available at the NCBI GEO website under GSE122782.RPB2: Yeast cells containing a His6-Tev-Biotin (HTB)-tagged RPB2 allele in the genome were used for this experiment. Cell pellets were prepared, lysed and sonicated similar to Sae2 ChIP, except the following lysis buffer was used - 25 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 0.1% NP40, 0.1% SDS, 1% Triton, 200 mM PMSF supplemented with protease inhibitors . After sonication the lysate was divided into three parts - 10% for Input, 45% for Rpb2 IP and 45% for mock IP. The IP was done with streptavidin linked M280 magnetic beads (Invitrogen) overnight and sheep anti-mouse M280 magnetic beads (Invitrogen) were used for the mock. The next day the beads were separated and washed 3 times with the following buffers - 1 , 2 , 3 , and 4 . For each wash, the beads were rotated for 5 min at RT. The fold enrichment over Input at different genomic loci was calculated using SYBR-Green based quantitative PCR. Samples were tested at various dilution factors to optimize a dilution factor for a CT value in the linear range. Then percentages of DNA enrichment of IP over the Input were calculated by using the following equation:(input)-CT(IP)-log - 100%*2^(CT(input)-CT(Mock IP)-log).-tagged SEN1 allele in the genome were prepared, lysed and sonicated similar to RPB2 ChIP except that all samples were prepared with CPT exposure (100 μM camptothecin or DMSO for 50 min). The IP was performed with streptavidin linked M280 magnetic beads (Invitrogen). The fold enrichment over Input at different genomic loci was calculated using SYBR-Green based quantitative PCR. Samples were tested at various dilution factors to optimize a dilution factor for a CT value in the linear range. Then percentages of DNA enrichment of IP over the Input were calculated by using the following equation: (100%*2^(CT(input)-CT(IP)-log). The relative fold of enrichment was calculated by normalizing the percentage of DNA enrichment of IP of each group to WT cells.Input - Log - CtDRIP)Yeast cells were grown as described for the Sae2 ChIP experiment, except there were no G2 cell samples and no formaldehyde crosslinking was done. DRIP was performed as previously described ; a detaiPrimer sequences used in this study were adapted from  and are For each sample, overlap between peaks identified from ChIP-seq data and the top 10% of highly transcribed genes in yeast  Nagalak was asseMammalian cell culture: Human U2OS and HEK-293T cells were grown and maintained in tetracycline-free DMEM containing 10% FBS media in a humidified 37°C incubator in the presence of 5% CO2. All cell lines were treated and maintained in plasmocin (InVivoGen) to ensure no mycoplasma contamination.Invitrogen Gateway pENTR223 donor vector for hSenataxin(∆1–1850) was obtained from DNASU (#HsCD00505781), and cloned into pcDNA5/FRT/TO (ThermoFisher) vector to make pTP3531. shRNA CtIP was custom-made by Cellecta (pRSITEP--U6Tet-(sh)-EF1-TetRep-2A-shRNA-CtIP) and includes the shRNA expression cassette 5ʹ-GAGCAGACCTTTCTTAGTATAGTTAATATTCATAGCTATACTGAGAAAGGE. coli) containing an NLS and fused to mCherry (pTP3492) was generated from pcDNA5/FRT/TO (Invitrogen) and pICE-RNaseHI-mCherry . pcDNA5 with NLS-mCherry (pTP3494) was generated from pcDNA5/FRT/TO (invitrogen) and pICE-mCherry (Addgene # 60364). pcDNA5-RNaseHI-D10R-E48R-NLS-mCherry (pTP3493) was cloned using pcDNA5/FRO/TO and pICE-RNaseHID10R-E48R-NLS-mCherry  was cloned using pLenti PGK GFP Blast (w510-5) which was a gift from Eric Campeau and Paul Kaufman (Addgene plasmid #19069 eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your work entitled "Sae2/CtIP prevents R-loop accumulation in eukaryotic cells" for consideration by eLife at this moment. We believe that this is very interesting work with exciting results, but it needs further analysis to support the model. However, we would be happy to consider a resubmission of your work, if you can appropriately respond to the reviewers.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in sae2∆ mutants such as CPT sensitivity and R-loop accumulation in the rDNA, authors conclude that yeast Sae2 is important for R-loop removal. They go on to show that Sae2 is found at sites of high transcription, especially upon treatment with CPT in S phase, and that its deletion leads to transcription-dependent damage sensitivity and Pol II stalling following CPT. Notably, most of these findings also apply for CtIP function in human cells. CtIP depletion triggers sensitivity to CPT that is rescued by Senataxin (the Sen1 human ortholog) overexpression. They find that RNA-DNA hybrids accumulate upon CTIP-loss or XPG-loss at laser-induced damage, based on binding of an mCherry-RNaseH sensor. Moreover, CtIP depletion decreases ssDNA break, suggesting that, as XPG, CtIP contributes to R-loop dissolution by generating a ssDNA break promoting the activity of senataxin and other hybrids helicases. The authors show that CtIP and XPG reduce breaks formed by CPT using both comet assays and LM-PCR. The most puzzling data came from combining both XPG and CtIP depletion, since, although their co-depletion completely abolishes the generation of an ssDNA break, no R-loop accumulate in these conditions. The authors interpret these data, as a lack of R-loop extension in these conditions, that would make them undetectable. Together, these studies lead the authors to propose that XPG and CtIP work in a redundant manner to process R-loops by generating a break at the R-loop site and allowing access of SETX to the hybrids for hybrid resolution. They propose a model by which CtIP would promote cleavage of Flap structures as a step to resolve R-loops.As I said, this is an interesting study performed in yeast and human cells to show that CtIP is involved in resolving R-loops in conjunction with XPG. By first observing the ability of Sen1 DNA-RNA helicase, involved in transcription termination, to suppress some of the phenotypes of The work is very interesting and exciting and provides an intriguing model and important function of CTIP to understand. However, additional results are needed to test the model and rule out other possibilities. Several experiments require additional control; all studies with S9.6 should provide the control with RNaseH treatment in vitro to show that the signal is removed . Although authors use Sen1 to remove R-loops, the data suggest that the terminator function of Sen1 has a role in the suppression of the CPT sensitivity and by extension the R-loops. In addition, the yeast data do not clearly support that RNAPII transcription is the major cause of R-loops. The authors are unable to show hybrids in RNAPII genes, but only in rDNA regions. Of particular note, the authors did not sufficiently establish the proposed order of events which place CtIP and XPG upstream of SETX. Is CtIP or XPG needed for Sen1/SETX to bind the DNA at the sites of hybrid formation, tested using the RNaseH sensor and ChIP assays? This would be one prediction of the model if nuclease processing is needed to generate access for Sen1/SETX. Could indeed the authors rule out that SETX and CtIP are not simply acting to reduce the same hybrid structures ? In summary, as it stands the authors need more data to substantiate their conclusions.Reviewer #1:sae2∆ mutants such as CPT sensitivity and R-loops detected by a specific antibody, authors conclude that yeast Sae2 is important for R-loop removal. The study is extended to human cells, in which using a parallel approach the authors conclude that CtIP have similar role. In this case, they use laser micro-irradiation and siXPG cells, which allow the authors to propose a model by which CtIP would be able to cleave Flap structures as a step to resolve R-loops. This is a new and a priori unexpected involvement of a DSB processing factor in R-loop removal that merits being considered for publication. However, the study has a number of issues that need to be resolved before it can be published. There are three important points that need to be solved first, apart from the essential experiments suggested below.This is an interesting study performed in yeast and human cells to show that CtIP is involved in resolving R-loops. By first observing the ability of Sen1 DNA-RNA helicase to suppress some of the phenotypes of - All studies with S9.6, except one in Figure 6A, did not provide the control with RNase H1 to show that the signal is removed. It is known that S9.6 also recognizes dsRNA and it is not possible to make conclusions with this antibody without removing the signal with RNase H1.- Although authors use Sen1 to remove R-loops, the data suggest that the terminator function of Sen1 has a role in the suppression of the CPT sensitivity and by extension the R-loops. It is necessary to show that RNH1 overexpression, not just Sen1/SETX also suppresses the CPT phenotype in yeast and that of micro-irradiated human cells.- Authors propose that CtIP/Sae2 may have a new FLAP endonucleolytic function similar to XPG. This conclusion is based on the similar phenotypes observed for CtIP and XPG-depleted cells. However, a similar phenotype may be produced by different mechanisms, and R-loop dynamics may respond to many different factors. The in vivo data showing that in the double depletion in human cells they have equal amount of R-loops than the simple is inconsistent with a model implying that an ssDNA nick (as that caused by S1 in vitro experiment) stabilizes R-loops. The in vivo data do not seem to support the model proposed since if CtIP is not able to cleaves the FLAP structure, R-loops would not be stabilized. In this context, the S1 experiment is really confusing and unnecessary in this study .- Provided the know function of CtIP in DSB resection and the relevance of transcription-replication collisions as a way to produce stalled and collapsed forks and considering the possibility that it could also lead to fork reversal generating a one-ended DSB-like structure susceptible of being resected by CtIP to restart the fork, it may be possible that the inability of CtIP to act at broken or reversal replication forks stalled in front of an R-loop lead to an accumulation of these hybrids as structures causing the lethality in CPT or microirradiation and Sae2/CtIP cells. Consequently, it is necessary to show that in vitro CtIP has the suggested activity. The assay done with S1 should indeed be performed with CtIP.sae2∆ strain.In Figure 1, authors should show overexpression of RNaseH to demonstrate that R-loops are the cause of CPT sensitivity in sae2∆ sensitivity to HU. Could it be transcription/replication collisions not R-loop dependent?Since the CPT sensitivity phenotype is observed in S-phase, it would be important to address if Sen1 and RNaseH1 rescue A DRIP-qPCR analysis of some of the genes with increased Sae2 recruitment in the ChIP-seq to validate the data.In Figure 1E, the control DRIP samples with RNaseH1 treatment is not shown.No statistical analysis is supplied for any of the yeast tables. This is essential in Figure 3A, where authors claim that o/e SEN1 reduces RNA Pol II accumulation. Again, an RNaseH1 o/e should be included.In Figure 4A, authors should show overexpression of RNaseH as a way to demonstrate that R-loops are the cause of CPT sensitivity in cells depleted of CtIP.Immunofluorescences in Figure 5C should have RNase H -treated cells as control. Image contrast could be improved and also show the channels separately.How do authors explain the reduction on R-loops compare to siC levels when both nucleases CtIP and XPG are missing? This is odd.Regarding the ssDNA breaks assay, due to the CtIP role in resection it is expected to have less breaks after DNA damage. Authors could perform the assay in a background known to increase R-loops, such as sen1 or rnh1 rnh2 and see if CtIP depletion reduces the R-loop-dependent DNA breaks.sae2∆ cells.The yeast data do not seem to support that RNAPII transcription is the major cause of R-loops. The authors are unable to show hybrids in RNAPII genes, but only in rDNA regions. Indeed, it makes sense from the data that CPT, that it is the rDNA region, in which the Tollervey lab has shown the relevance of Top1 to prevent R-loop formation, where the major intermediate leading to lethality may accumulate after CPT. Is there any data showing an involvement of Sen1 in RNAPI transcription termination? This needs to be considered, since at the end it is not really clear which function of Sen1 is able to suppress the sensitivity to CPT of The enrichment of Sae2 at 176 sites of transcription in S phase may not necessarily related with R-loops. Authors do not show whether such enrichment is reduced by RNase H overexpression. At least should be done by DRIP-qPCR to validate the data using RNase H.sae2∆ strains. It is required to reduce the signal by overexpressing RNH1. Overexpression of Sen1 produces a reduction in signal, but this may be due to the terminator function of Sen1. For this reason, authors need to revert the phenotype with a protein not involved in termination, but just in R-loop removal, such as RNH1.In the genome-wide analysis, the fact that RNAPII is found at high levels at sites previously described to from R-loops does not implies that R-loops are accumulated in I am not sure what the results of Figure 4B really says. If CPT causes breaks, it should not be surprising that CtIP increases the sensitivity. Provided the impact of transcription in collisions or increase in damage by other ways, the action of DRB should not be surprising. However, the reduction of the sensitivity phenotype is very small. Results could be repeated using another inhibitor of transcription such as α-amanitin.In Figure 5 it is necessary to show that all signals are reduced by RNase H.Reviewer #2:In this manuscript, Makharashvili et al., report a novel function for Sae2/CtIP in both yeast and human cells: They propose that this endonuclease, known to promote resection during DSB repair, also contributes in R-loops (DNA-RNA hybrids) dissolution, in conjunction with XPG. First, they found that yeast sen1, an RNA-DNA helicase involved in transcription termination, as well as transcription inhibitors, rescue the CPT sensitivity of sae2 deficient strains. They further report that Sae2 binds to highly transcribed genes following exposure to CPT in S phase, that it correlates with accumulation (pausing) of RNA Polymerase II at these positions in CPT treated cells, and that R-loops accumulate in Sae2 deficient strain (at the rDNA locus). Notably, most of these findings also apply for CtIP function in human cells: CtIP depletion triggers sensitivity to CPT that is rescued by Senataxin (the Sen1 human ortholog) overexpression and R-loops accumulate in CtIP-depleted cells, as they do in XPG-depleted cells, as previously reported. Moreover, CtIP depletion decreases ssDNA break, suggesting that, as XPG, CtIP contributes to R-loop dissolution by generating a ssDNA break promoting the activity of senataxin and other hybrids helicases. All these data identify Sae2/CtIP as a key regulator of R-loops stability on yeast and human genome. The most puzzling data came from combining both XPG and CtIP depletion, since, although their co depletion completely abolishes the generation of an ssDNA break, no R-loop accumulate in these conditions. The authors interpret these data, as a lack of R-loop extension in these conditions, that would make them undetectable.Given the threat of R-loop on genome stability that has been clearly described recently, identifying the mechanisms that function to regulate their occurrence on the genome is of prime interest. Additionally, the model presented here is very interesting and exciting. Yet I feel that (i) the model would be more convincing if the same loci were analyzed across the Figures, (see main point 1 below) and (ii) some key experiment are lacking to fully support the model (see main point 2).For the yeast part, the authors need to show that indeed, R-loops accumulates at sites that recruit sae2 upon CPT treatment. They show Figure 2E that they do accumulate on rDNA, but to be consistent with Figure 2C, and Figure 3A-B and to demonstrate convincingly the relationship at specific loci between Sae2 and R-loops, the authors need to report the accumulation of R-loops at ENO1, ADH2, FBA1, as well as SNR13, SNR5. Additionally, the authors need to extend their Sae2-flag ChIP-seq data analysis more specifically by comparing their data with DRIP-seq yeast genome profiles .For the mammalian cells part, the fact that codepletion of XPG and CtIP shows no R-loops accumulation but deficient ssDNA breakage is really puzzling. The authors propose that the hybrids still accumulate in these conditions but are not detectable using their assay (S9.6 and mutated cherry RNAseH). The author needs to demonstrate that this is the case. Indeed, that CtIP depletion rescues R-loop accumulation in XPG-depleted cells may also indicate that CtIP lies downstream XPG mediated-ssDNA cleavage, hence challenging their model. I realize this may be difficult to assess, but this is an important piece of data that is lacking to support their current model; maybe the authors could try at one locus non denaturing sodium bisulfite treatment to detect ssDNA ?Figure 1: Sen 1 also rescues viability upon CPT in mre 11 deficient strain. Can the author comment and integrate the MRX complex in their R-loop processing model?Figure 2: The ChIP-seq data should be more described, and a full supplemental Figure should be provided. Do the 45 peaks also overlap with the 176 peaks in presence of CPT? If yes, can we see some examples on browser? Can the author show a scatter plot where for each gene, the transcription level is plotted against the CPT-treated enrichment of Sae2 ? The author could also retrieve RNA Pol II mapping and perform the same analysis. Another very common way to show this type of data, would be to separate the yeast genes based on their expression level and show that Sae2 binding is statistically higher for the highly expressed genes subset compared to the low expressed set of genes. Finally, Sae2 binding in S phase upon CPT should be compared to DRIP-seq data from the Koshland lab . Given that the authors show R-loop accumulation at rDNA, can they also report the change of Sae2 recruitment upon CPT on rDNA from their Sae2 ChIP-seq? Can we also see the snapshot for PDC1, SNR13, SNR5 for Sae2 distribution in CPT treated samples?Figure 3: "We did not observe CPT induced RNA Pol II pausing at PDC1". It seems from the Figure that the author can actually see some Rbp2 increase .Figure 5: In their expression study, is there some overlap between genes modified following CPT treatment and genes modified after CtIP depletion? Can the author directly show the DRIP-seq count on the different gene subsets  compared to random genes ?Figure 6: Part of the Figure 6B, can the author show the R-loop level by DRIP qPCR (only at the b-actin gene) upon XPG and CtIP depletion?Figure 8: the assay presented Figure 8D is very nice. Could the author also perform XPG only and CtIP/XPG depletion to further validate that indeed ssDNA breakage is absent in these conditions?Reviewer #3:In this manuscript, the role of Sae2/CTIP in R-loop accumulation and processing is explored both in yeast and mammalian cells. Motivated by understanding the unexpected sensitivity of Sae2/CTIP-deficient cells to CPT-induced 3'-ssDNA lesions, Paull and colleagues explored the role of transcriptional regulation in survival of Sae2-deficient cells. Upon finding that SEN1 expression rescues this sensitivity and that its loss enhances sensitivity, they go on to show that Sae2 is found at sites of high transcription, especially upon treatment with CPT, and that its deletion leads to transcription-dependent damage sensitivity and Pol II stalling following CPT. Similarly, they find in mammalian cells that sensitivity due to CTIP loss can be rescued by SETX expression and that RNA-DNA hybrids accumulate upon CTIP-loss or XPG-loss at laser-induced damage, based on binding of an mCherry-RNaseH sensor. Hybrids are also elevated in the absence of damage and these nucleases both at genes know to form R-loops and R-loop inducible sites. Finally, the authors show that CtIP and XPG reduce breaks formed by CPT using both comet assays and LM-PCR. Together, these studies lead the authors to propose that XPG and CtIP work in a redundant manner to process R-loops by generating a break at the R-loop site and allowing access of SETX to the hybrids for hybrid resolution.While this is an intriguing model and important function of CTIP to understand, I think that the model is somewhat premature at this point and additional results are needed to test the model and rule out other possibilities. Several experiments require additional controls as well. Of particular note, I don't think the authors have sufficiently established the proposed order of events which place CtIP and XPG upstream of SETX. Is CtIP or XPG needed for Sen1/SETX to bind the DNA at the sites of hybrid formation, tested using the RNaseH sensor and ChIP assays? This would be one prediction of the model if nuclease processing is needed to generate access for Sen1/SETX. Also, can the authors rule out that SETX and CtIP are not simply acting to reduce the same hybrid structures ? As it stands the authors need more data to substantiate their conclusions.Where does the CPT-induced break fit into the authors' model? The break induced by CtIP/XPG would be in addition to this CPT-induced break. This is omitted from Figures and models and a somewhat confusing point.Figure 2: As a specificity control the authors should test whether Sae2 binding by ChIP is blocked by transcription inhibition.Figure 2: Since the DRIP did not work well in yeast, it would be helpful to show that Sae2 binding by ChIP is rescued by overexpression of RNaseH or Sen1 in yeast cells. Similarly, they could ask if RNaseH overexpression rescues CPT effects on survival.Figure 3: The authors need to analyze several other genes that are not expressed or expressed at lower levels to show specificity of the effect of CtIP loss at highly expressed genes. Right now they examine two genes, PDC1 and NRD1 as negative controls (if I understand this correctly) and Pol II binding is actually elevated at the first site.Figure 4: Does RNaseH overexpression rescue viability of CTIP deletion? Also, please comment why in Figure 4 hybrids increase with DRB treatment in WT cells?Figure 4, Figure 5 and Figure 6: Is the nuclease activity of XPG required for any of the observed effects? Testing this in at least one of the assays would be helpful.Figure 5D: Does RNaseH expression in vivo and RNaseH treatment in vitro reduce the increased hybrid signal . This is an important control.Figure 5E/F: What is the rationale for both increases and decreases in transcription overlapping with site of hybrid formation? I don't understand the implications of the RNA-seq data. Also, Figure 5F is missing in the Figure, making these data difficult to evaluate.Figure 5: What is the impact of CtIP and XPG loss, or loss of both, on the cell cycle. Are breaks or hybrid levels reduced due to alterations in cell cycle progression (e.g. failure to enter S phase) or accumulation in G2. It seems in WT cells hybrids are naturally higher in G2 and if breaks depend on S phase entry changes in cell cycle could affect the interpretation.Figure 6: Why are there no more breaks upon addition of CPT? Shouldn't the CPT itself lead to break formation even without the action of XPG and CtIP? Also, alkaline comet is not specific for SSB detection – these could be DSBs. I suggest the authors use caution in the way they present these data.The idea that the R-loops are too small to be detected when both XPG and CtIP are lost is not well supported. Isn't it possible that there is no R-loop under these conditions and that RNAPII has simply stalled and is the toxic lesion? Can RNaseH expression rescue sensitivity due to loss of both XPG and CtIP? That is one test of this idea. Or can evidence for a smaller R-loops be obtained using bisulfite sequencing. Minimally I think other models should be considered.[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your article has been reviewed by the same three peer reviewers that revised the first submission, and the evaluation has been overseen by the same Reviewing Editor and Jessica Tyler as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Sae2/CtIP prevents R-loop accumulation in eukaryotic cells" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Makharashvili et al., report a novel function for Sae2/CtIP in both yeast and human cells, and propose that Sae2/CtIP, known to promote resection during DSB repair, also contributes in R-loop (DNA-RNA hybrids) dissolution, in conjunction with XPG. In this revised version, the authors have addressed to a reasonable extent my main points. More specifically they are now consistent throughout the manuscript, and report Sae2 binding and R-loop accumulation in Sae2 deleted strain at the same loci. They have also now changed their discussion to take more hypotheses into account. They have also largely controlled their data by performing RNAseH treatment to validate their results obtained with S9.6 antibody. This manuscript is a significantly improved version of the previous one. Results are now clearer and convincing. However, the key conclusion of their model that CtIP cleaves the loop needs further support and clarification before acceptance.Essential revisions:The results on which the model proposed rely on CPT-treated cells  (CPT creates ssDNA breaks with Top1-cc) or cells upon laser-induced breaks . So, the experimental conditions used implies to start with a genotoxic-induced break. It is still therefore unclear why CtIP would be required for an additional break. Indeed, authors were not able to show the proposed activity of CtIP in vitro. Authors would need to show the action of CtIP in untreated cells , with no break, to support their model that demands that CtIP cleaves the loop. The known action of the Sae2/CtIP nuclease activity on DNA resection as a requirement to remove the RNA-DNA hybrid accumulated at a break would make much more sense and fit better with the combined effect with XPG. Therefore, either authors provide new data with untreated cells to support their model as such or alternatively include in their model the breaks caused by CPT/laser irradiation to explain the role of CtIP under such conditions. [Editors’ note: the author responses to the first round of peer review follow.]As I said, this is an interesting study performed in yeast and human cells to show that CtIP is involved in resolving R-loops in conjunction with XPG. By first observing the ability of Sen1 DNA-RNA helicase, involved in transcription termination, to suppress some of the phenotypes of sae2∆ mutants such as CPT sensitivity and R-loop accumulation in the rDNA, authors conclude that yeast Sae2 is important for R-loop removal. They go on to show that Sae2 is found at sites of high transcription, especially upon treatment with CPT in S phase, and that its deletion leads to transcription-dependent damage sensitivity and Pol II stalling following CPT. Notably, most of these findings also apply for CtIP function in human cells. CtIP depletion triggers sensitivity to CPT that is rescued by Senataxin (the Sen1 human ortholog) overexpression. They find that RNA-DNA hybrids accumulate upon CTIP-loss or XPG-loss at laser-induced damage, based on binding of an mCherry-RNaseH sensor. Moreover, CtIP depletion decreases ssDNA break, suggesting that, as XPG, CtIP contributes to R-loop dissolution by generating a ssDNA break promoting the activity of senataxin and other hybrids helicases. The authors show that CtIP and XPG reduce breaks formed by CPT using both comet assays and LM-PCR. The most puzzling data came from combining both XPG and CtIP depletion, since, although their co-depletion completely abolishes the generation of an ssDNA break, no R-loop accumulate in these conditions. The authors interpret these data, as a lack of R-loop extension in these conditions, that would make them undetectable. Together, these studies lead the authors to propose that XPG and CtIP work in a redundant manner to process R-loops by generating a break at the R-loop site and allowing access of SETX to the hybrids for hybrid resolution. They propose a model by which CtIP would promote cleavage of Flap structures as a step to resolve R-loops.The work is very interesting and exciting and provides an intriguing model and important function of CTIP to understand. However, additional results are needed to test the model and rule out other possibilities. Several experiments require additional control; all studies with S9.6 should provide the control with RNaseH treatment in vitro to show that the signal is removed . Although authors use Sen1 to remove R-loops, the data suggest that the terminator function of Sen1 has a role in the suppression of the CPT sensitivity and by extension the R-loops. In addition, the yeast data do not clearly support that RNAPII transcription is the major cause of R-loops. The authors are unable to show hybrids in RNAPII genes, but only in rDNA regions. Of particular note, the authors did not sufficiently establish the proposed order of events which place CtIP and XPG upstream of SETX. Is CtIP or XPG needed for Sen1/SETX to bind the DNA at the sites of hybrid formation, tested using the RNaseH sensor and ChIP assays? This would be one prediction of the model if nuclease processing is needed to generate access for Sen1/SETX. Could indeed the authors rule out that SETX and CtIP are not simply acting to reduce the same hybrid structures ? In summary, as it stands the authors need more data to substantiate their conclusions.Thank you for the in-depth comments. From the summary we received, it seems that the major issues have to do with: (1) confirmation of the specificity of S9.6 immunoprecipitations, (2) demonstration of the ability of RNaseH suppression in addition to SEN1/Senataxin suppression of CtIP depletion phenotypes, (3) demonstration of R-loops at RNAPII genes in yeast, and (4) further investigation and validation of the ideas in our proposed model. We have performed several additional experiments to address these points, as discussed below. We think the manuscript is significantly improved with these revisions and additions to the data. We have also revised our thinking about the model and have incorporated some alternative scenarios into the discussion in the main text. We address these four main questions first and then answer the reviewer questions below.1) RNaseH in vitro treatment for validation of S9.6 IPs:We repeated the S9.6 DRIP-qPCR experiment using pre-treatment with RNaseH in vitro as a control. These results, shown in Figure 6C, and Figure 6—Figure supplement 3 that the S9.6 signal is in fact due to RNA-DNA hybrids as it is eliminated by RNaseH.2) Another suggestion of the reviewers was to show RNaseH suppression of phenotypes in yeast and in human cells, which would confirm the R-loop-related role of SEN1/SETX.Δsae2 yeast cells to camptothecin and to MMS, as shown in Figure 1.Galactose-induced overexpression of RNH1 also suppresses the sensitivity of We also performed this experiment in human cells by overexpressing RNaseH in cells depleted of CtIP. We found that this treatment also reduces R-loops in CtIP-depleted cells, as measured by DRIP-qPCR. This result is now shown in Figure 6B and D.sae2∆ yeast cells? To answer this question we re-visited DRIP experiments in yeast. We observed a statistically significant increase in R-loops at highly transcribed loci in sae2∆ cells, as in Figure 2E .3) Is RNAPII transcription the cause of R-loop accumulation in 4) To address whether the proposed model of SEN1/SETX recruitment to transcription sites via Sae2/CtIP is correct.We decided to determine if SEN1 and SETX recruitment to relevant sites in both yeast and human cells is Sae2/CtIP-dependent. In yeast, we used an HTB-tagged SEN1 strain and examined recruitment to several of the loci where we observed high levels of Sae2 protein and also R-loop accumulation. This experiment shows that SEN1 is actually present at higher levels than in wild-type strains  .In human cells we performed ChIP-qPCR experiments to determine if endogenous Senataxin is present at sites of R-loop accumulation. We used our inducible Sγ3 locus for these experiments since we are able to control the transcription at this site. Similar to the experiment in yeast, we found that Senataxin occupancy is actually higher in CtIP-depleted cells than in wild-type, and that this is dependent on active transcription . It was also higher with XPG depletion but the p value for this comparison was 0.06.sae2∆ or CtIP-depleted cells because this alternate pathway is augmented by the increased removal of RNA-DNA hybrids. We now present both of these models in the main text and in Figure 7.We conclude from this that the recruitment of SEN1/SETX protein is not impaired, yet there seems to be a significant dysfunction of the enzyme since levels of R-loops are high and RNAPII is found to be stalled at these sites. One possibility is that SEN1/SETX recruitment to the site is mediated by association with the polymerase or other factors, yet the enzyme is not able to access the R-loop efficiently because of a problem with the structure of the DNA at the locus . It is also possible that Sae2/CtIP is in a separate, parallel pathway to SEN1/SETX and that SEN1 overexpression (or RNaseH overexpression) helps in Reviewer #1:This is an interesting study performed in yeast and human cells to show that CtIP is involved in resolving R-loops. By first observing the ability of Sen1 DNA-RNA helicase to suppress some of the phenotypes of sae2∆ mutants such as CPT sensitivity and R-loops detected by a specific antibody, authors conclude that yeast Sae2 is important for R-loop removal. The study is extended to human cells, in which using a parallel approach the authors conclude that CtIP have similar role. In this case, they use laser micro-irradiation and siXPG cells, which allow the authors to propose a model by which CtIP would be able to cleave Flap structures as a step to resolve R-loops. This is a new and a priori unexpected involvement of a DSB processing factor in R-loop removal that merits being considered for publication. However, the study has a number of issues that need to be resolved before it can be published. There are three important points that need to be solved first, apart from the essential experiments suggested below.1) All studies with S9.6, except one in Figure 6A, did not provide the control with RNase H1 to show that the signal is removed. It is known that S9.6 also recognizes dsRNA and it is not possible to make conclusions with this antibody without removing the signal with RNase H1.We have repeated the DRIP-qPCR experiments and performed RNaseH treatment of the samples before the IP , showing that the signal is specific to RNA-DNA hybrids.- Although authors use Sen1 to remove R-loops, the data suggest that the terminator function of Sen1 has a role in the suppression of the CPT sensitivity and by extension the R-loops. It is necessary to show that RNH1 overexpression, not just Sen1/SETX also suppresses the CPT phenotype in yeast and that of micro-irradiated human cells.See Figure 1 and Figure 6B,D.- Authors propose that CtIP/Sae2 may have a new FLAP endonucleolytic function similar to XPG. This conclusion is based on the similar phenotypes observed for CtIP and XPG-depleted cells. However, a similar phenotype may be produced by different mechanisms, and R-loop dynamics may respond to many different factors. The in vivo data showing that in the double depletion in human cells they have equal amount of R-loops than the simple is inconsistent with a model implying that an ssDNA nick (as that caused by S1 in vitro experiment) stabilizes R-loops. The in vivo data do not seem to support the model proposed since if CtIP is not able to cleaves the FLAP structure, R-loops would not be stabilized. In this context, the S1 experiment is really confusing and unnecessary in this study .We find that the double depletion of both CtIP and XPG generates a very low level of R-loops, similar to wild-type cells, whereas each single depletion shows high R-loops. So, the double depletion is not equivalent to either of the single depletions. To explain this, we propose that extensive R-loops are not being formed in the double depletion cells because of a lack of single-strand processing. The Lieber laboratory showed previously that a nick in either strand strongly promotes stabilization of R-loops, so our hypothesis is consistent with this idea. We agree that we don't need to show the nick inducing R-loops since this was published previously.The model is simply a diagrammatic set of working hypotheses that we are using to frame questions around this data; we are certainly not claiming to have proven everything in the model, and the discussion is modified now to better reflect this view. We know that CtIP and XPG are not completely redundant because we see obvious R-loop accumulation in the absence of either factor. We are working toward understanding the differences between their activities using purified components, but this is beyond the scope of the work being considered here.- Provided the know function of CtIP in DSB resection and the relevance of transcription-replication collisions as a way to produce stalled and collapsed forks and considering the possibility that it could also lead to fork reversal generating a one-ended DSB-like structure susceptible of being resected by CtIP to restart the fork, it may be possible that the inability of CtIP to act at broken or reversal replication forks stalled in front of an R-loop lead to an accumulation of these hybrids as structures causing the lethality in CPT or microirradiation and Sae2/CtIP cells. Consequently, it is necessary to show that in vitro CtIP has the suggested activity. The assay done with S1 should indeed be performed with CtIP.We have made R-loops in vitro using prokaryotic and phage RNA polymerases but have not observed significant levels of processing of these structures. Considering the known association between CtIP and human transcription-associated complexes though, perhaps it is not surprising that a reconstitution with heterologous polymerase would not work here.It is certainly possible that some of the consequences of CtIP depletion involve fork reversal. We now have alleles of CtIP that can separate the function of CtIP in canonical DSB processing from the nuclease-related function, so we are in the process of testing these.In Figure 1, authors should show overexpression of RNaseH to demonstrate that R-loops are the cause of CPT sensitivity in sae2∆ strain.∆sae2 strain. We also show that RNaseH overexpression in human U2OS cells reduces R-loops in CtIP-depleted cells .As shown in Figure 1, we do observe suppression of CPT and MMS sensitivity by RNH1 in a Since the CPT sensitivity phenotype is observed in S-phase, it would be important to address if Sen1 and RNaseH1 rescue sae2∆ sensitivity to HU. Could it be transcription/replication collisions not R-loop dependent?sae2 strains to hydroxyurea with either RNH1 or SEN1 overexpression and found no evidence for suppression of sensitivity under these conditions  shows that the role of RNA removal is critical.p values have been added to Figure 3. We do not have RNaseH1 overexpression in this case but the fact that RNH1 overexpression rescues growth of In Figure 4A, authors should show overexpression of RNaseH as a way to demonstrate that R-loops are the cause of CPT sensitivity in cells depleted of CtIP.We now show this in Figure 1 in yeast as well as in Figure 6 in human cells.Immunofluorescences in Figure 5C should have RNase H -treated cells as control. Image contrast could be improved and also show the channels separately.We expressed RNaseH in cells with CtIP depletion, as shown in How do authors explain the reduction on R-loops compare to siC levels when both nucleases CtIP and XPG are missing? This is odd.The idea is that nicks in DNA promote the extension and stabilization of R-loops. This has been shown in vitro by Lieber and colleagues and we have confirmed this result . In the absence of a single-strand break, RNA-DNA hybrids are extremely constrained topologically, and the region of the RNA-DNA hybrid is likely to be inaccessible because of the presence of the non-template strand of DNA. So, we are proposing that, in the absence of strand breaks, there is a nascent structure that is not efficiently recognized by S9.6 or RNaseH but yet is toxic to cells and needs to be removed. We speculate that this could be a small R-loop or a paused polymerase associated with a small R-loop but do not know exactly what this structure is.Regarding the ssDNA breaks assay, due to the CtIP role in resection it is expected to have less breaks after DNA damage. Authors could perform the assay in a background known to increase R-loops, such as sen1 or rnh1 rnh2 and see if CtIP depletion reduces the R-loop-dependent DNA breaks.Actually, we would expect more breaks in the absence of CtIP if its role is primarily in double-strand break repair because presumably there would be more unresolved breaks. In the manuscript we are using an alkaline comet assay which measures single-strand breaks, and here we always observe fewer in the absence of CtIP. Resection of double-strand breaks would not be expected to change the total number of single-strand breaks. Unfortunately, we do not have any method to measure single-strand breaks in yeast as the reviewer suggests. In human cells we have measured single-strand breaks in XPG-depleted cells (which have higher R-loops), and in this context, removal of CtIP also lowers the number of single-strand breaks .The yeast data do not seem to support that RNAPII transcription is the major cause of R-loops. The authors are unable to show hybrids in RNAPII genes, but only in rDNA regions. Indeed, it makes sense from the data that CPT, that it is the rDNA region, in which the Tollervey lab has shown the relevance of Top1 to prevent R-loop formation, where the major intermediate leading to lethality may accumulate after CPT. Is there any data showing an involvement of Sen1 in RNAPI transcription termination? This needs to be considered, since at the end it is not really clear which function of Sen1 is able to suppress the sensitivity to CPT of sae2∆ cells.sae2∆ strains with DNA damage. Sen1 is definitely involved in transcription termination, of non-coding RNAs as well as a subset of RNAPII-transcribed protein coding genes (1–3). We are not trying to eliminate termination as part of the possible mechanism here; in fact, we also show in Figure 1 the ability of PCF11 to partially suppress sae2 DNA damage sensitivity, and PCF11 is primarily known as a transcription termination factor (4). PCF11 also promotes the association of SEN1 with RNAPII however, and both sen1 and pcf11 mutations in yeast induce higher R-loops (4). It is also important to point out that SEN1 promotes transcription termination by removal of nascent RNA made by RNA Pol I as well as by RNA Pol II (5), although we removed the rDNA data here is favor of the RNAPII data.See point #3 above. We do now show higher levels of R-loops at RNAPII genes in The enrichment of Sae2 at 176 sites of transcription in S phase may not necessarily related with R-loops. Authors do not show whether such enrichment is reduced by RNase H overexpression. At least should be done by DRIP-qPCR to validate the data using RNase H.sae2∆ strains, and we show RNaseH treatment of the S9.6 IPs eliminates the signal. We also show that RNH1 expression reduces the sensitivity of sae2∆ strains to both CPT and MMS, and we show that RNaseH overexpression in human cells reduces levels of R-loops in CtIP-depleted cells. We are pretty darn sure that these sites have R-loops. We are not saying that Sae2/CtIP is going to these sites because there are R-loops there, but that Sae2/CtIP goes to sites where high transcription is happening, and that in the absence of Sae2/CtIP, there are R-loops.We now show that the sites we isolated with high Sae2 occupancy do have higher R-loops in In the genome-wide analysis, the fact that RNAPII is found at high levels at sites previously described to from R-loops does not implies that R-loops are accumulated in sae2∆ strains. It is required to reduce the signal by overexpressing RNH1. Overexpression of Sen1 produces a reduction in signal, but this may be due to the terminator function of Sen1. For this reason, authors need to revert the phenotype with a protein not involved in termination, but just in R-loop removal, such as RNH1.Δsae2 strains to both CPT and MMS , and overexpression of wild-type RNaseH reduces levels of R-loops in CtIP-depleted human cells. Also, we found that the sen1-R302W allele rescues Δsae2 DNA damage sensitivity similar to wild-type SEN1. This mutation has been reported to abrogate the terminator function of SEN1 by blocking interaction of SEN1 with Rpb1 (6), suggesting that the terminator function of SEN1 is not critical . In contrast, mutation of the helicase domain does reduce the ability of SEN1 to suppress the phenotype, and a combination of this mutation with a Δsae2 deletion generates extreme DNA damage sensitivity .Overexpression of RNH1 does suppress the phenotype of I am not sure what the results of Figure 4B really says. If CPT causes breaks, it should not be surprising that CtIP increases the sensitivity. Provided the impact of transcription in collisions or increase in damage by other ways, the action of DRB should not be surprising. However, the reduction of the sensitivity phenotype is very small. Results could be repeated using another inhibitor of transcription such as α-amanitin.The CPT sensitivity assay in Figure 4B shows partial suppression of the sensitivity observed in CtIP-depleted cells by DRB treatment. The fact that we see partial restoration is expected because the transcription-associated role of CtIP that we are describing in this work is only part of its diverse biological functions, which also include promoting the nuclease activity of Mre11 at double-strand break sites. We would not expect transcription inhibition to necessarily affect this aspect of its function if the double-strand break is the critical lesion. We also previously tested α-amanitin but we found it was too toxic in this type of assay.In Figure 5 it is necessary to show that all signals are reduced by RNase H.See point #1 above.Reviewer #2:In this manuscript, Makharashvili et al., report a novel function for Sae2/CtIP in both yeast and human cells: They propose that this endonuclease, known to promote resection during DSB repair, also contributes in R-loops (DNA-RNA hybrids) dissolution, in conjunction with XPG. First, they found that yeast sen1, an RNA-DNA helicase involved in transcription termination, as well as transcription inhibitors, rescue the CPT sensitivity of sae2 deficient strains. They further report that Sae2 binds to highly transcribed genes following exposure to CPT in S phase, that it correlates with accumulation (pausing) of RNA Polymerase II at these positions in CPT treated cells, and that R-loops accumulate in Sae2 deficient strain (at the rDNA locus). Notably, most of these findings also apply for CtIP function in human cells: CtIP depletion triggers sensitivity to CPT that is rescued by Senataxin (the Sen1 human ortholog) overexpression and R-loops accumulate in CtIP-depleted cells, as they do in XPG-depleted cells, as previously reported. Moreover, CtIP depletion decreases ssDNA break, suggesting that, as XPG, CtIP contributes to R-loop dissolution by generating a ssDNA break promoting the activity of senataxin and other hybrids helicases. All these data identify Sae2/CtIP as a key regulator of R-loops stability on yeast and human genome. The most puzzling data came from combining both XPG and CtIP depletion, since, although their co depletion completely abolishes the generation of an ssDNA break, no R-loop accumulate in these conditions. The authors interpret these data, as a lack of R-loop extension in these conditions, that would make them undetectable.Given the threat of R-loop on genome stability that has been clearly described recently, identifying the mechanisms that function to regulate their occurrence on the genome is of prime interest. Additionally, the model presented here is very interesting and exciting. Yet I feel that (i) the model would be more convincing if the same loci were analyzed across the Figures, (see main point 1 below) and (ii) some key experiment are lacking to fully support the model (see main point 2).For the yeast part, the authors need to show that indeed, R-loops accumulates at sites that recruit sae2 upon CPT treatment. They show Figure 2E that they do accumulate on rDNA, but to be consistent with Figure 2C, and Figure 3A-B and to demonstrate convincingly the relationship at specific loci between Sae2 and R-loops, the authors need to report the accumulation of R-loops at ENO1, ADH2, FBA1, as well as SNR13, SNR5. Additionally, the authors need to extend their Sae2-flag ChIP-seq data analysis more specifically by comparing their data with DRIP-seq yeast genome profiles .See point #3 above, and Figure 2E. Comparison to DRIPseq data sets is discussed below.For the mammalian cells part, the fact that codepletion of XPG and CtIP shows no R-loops accumulation but deficient ssDNA breakage is really puzzling. The authors propose that the hybrids still accumulate in these conditions but are not detectable using their assay (S9.6 and mutated cherry RNAseH). The author need to demonstrate that this is the case. Indeed, that CtIP depletion rescues R-loop accumulation in XPG-depleted cells may also indicate that CtIP lies downstream XPG mediated-ssDNA cleavage, hence challenging their model. I realize this may be difficult to assess, but this is an important piece of data that is lacking to support their current model; maybe the authors could try at one locus non denaturing sodium bisulfite treatment to detect ssDNA ?Much more detailed analysis is required to determine exactly what the nature of the initiating lesion is. We know there is some type of lesion, since removal of both factors results in very high sensitivity to DNA damage. We do not think the data is consistent with CtIP creating a toxic intermediate downstream of XPG action because if this were the case, CtIP depletion would rescue the sensitivity of XPG-depleted cells and this is not the case. We propose that the lesion is a nascent R-loop but it could also be simply a stalled polymerase, if secondary structure in DNA were exposed in the stalled complex. Certainly, the nature of the lesion is of interest to us, and we are working on reconstituting this in vitro, but determining this conclusively is likely the topic of an entire subsequent study.Figure 1: Sen 1 also rescues viability upon CPT in mre 11 deficient strain. Can the author comment and integrate the MRX complex in their R-loop processing model?We are working on that but do not have enough data to speculate on what the role of MRX(N) is in R-loop processing.Figure 2: The ChIP-seq data should be more described, and a full supplemental Figure should be provided. Do the 45 peaks also overlap with the 176 peaks in presence of CPT? If yes, can we see some examples on browser? Can the author show a scatter plot where for each gene, the transcription level is plotted against the CPT-treated enrichment of Sae2 ? The author could also retrieve RNA Pol II mapping and perform the same analysis. Another very common way to show this type of data, would be to separate the yeast genes based on their expression level and show that Sae2 binding is statistically higher for the highly expressed genes subset compared to the low expressed set of genes.This is what we did in Figure 2D. We look at the overlap between the sites where Sae2 is bound in S phase with CPT and compare them to the top 10% of transcribed genes (ranked by expression level) versus the same number of randomly chosen genes. The comparison with the randomly chosen genes is done 1000 times, which generates a distribution. Only in the case of S phase plus CPT is there a statistically significant difference between the overlap with the top 10% of genes and the overlap with the randomly selected genes. More detailed information about the confidence intervals for the transcription analysis is now shown in Supplementary file 1. This shows that the p value for the S+CPT immunoprecipitation overlapping with the top 10% of transcribed genes is <.0053, whereas the analysis of the S phase sample with no DNA damage yield a p value <.757. We have not done in-depth analysis of transcription levels versus Sae2 enrichment because the transcription levels we are using are from published RNA-seq data, not the S phase or S phase plus CPT cells that we are using here, which would be preferable. We are working on doing this in mammalian cells with CtIP ChIP but this is still in progress.With respect to the comparison between the S phase peaks and the S plus CPT peaks, there are relatively few peaks that overlap. A browser view of a few of these overlapping genes is shown in Finally, Sae2 binding in S phase upon CPT should be compared to DRIP-seq data from the Koshland lab .9 of 45 Sae2 peaks in S phase (20%) and 36 of 176 Sae2 peaks in S phase with CPT (20%) overlap with the DRIP peaks in wild-type cells from Wahba et al., 2016. This is now stated in the text.Given that the authors show R-loop accumulation at rDNA, can they also report the change of Sae2 recruitment upon CPT on rDNA from their Sae2 ChIP-seq? Can we also see the snapshot for PDC1, SNR13, SNR5 for Sae2 distribution in CPT treated samples?There is substantial recruitment of Sae2 to the rDNA, which increases with CPT exposure, and we do observe Sae2 at PDC1 , but theFigure 3: "We did not observe CPT induced RNA Pol II pausing at PDC1". It seems from the Figure that the author can actually see some Rbp2 increase .We corrected the description in the text.Figure 5: In their expression study, is there some overlap between genes modified following CPT treatment and genes modified after CtIP depletion? Can the author directly show the DRIP-seq count on the different gene subsets  compared to random genes ?We did not do DRIP-seq. We did RNA-seq and compared the locations of genes with differences in gene expression depending on CtIP status and CPT exposure to previously published DRIPseq data . This analysis showed that the genes affected by CtIP loss are overrepresented in the DRIP-seq dataset (p<0.0052 for the comparison between – /+ CtIP and the genes identified by DRIPseq). The 99% confidence intervals have been added to the supplementary file as well as cited in the text.Figure 6: Part of the Figure 6B, can the author show the R-loop level by DRIP qPCR (only at the b-actin gene) upon XPG and CtIP depletion?We were not able to do this with the large number of experiments requested.Figure 8: The assay presented Figure 8D is very nice. Could the author also perform XPG only and CtIP/XPG depletion to further validate that indeed ssDNA breakage is absent in these conditions?We would like to do this but had to prioritize other experiments based on the summary of the reviews.Reviewer #3:In this manuscript, the role of Sae2/CTIP in R-loop accumulation and processing is explored both in yeast and mammalian cells. Motivated by understanding the unexpected sensitivity of Sae2/CTIP-deficient cells to CPT-induced 3'-ssDNA lesions, Paull and colleagues explored the role of transcriptional regulation in survival of Sae2-deficient cells. Upon finding that SEN1 expression rescues this sensitivity and that its loss enhances sensitivity, they go on to show that Sae2 is found at sites of high transcription, especially upon treatment with CPT, and that its deletion leads to transcription-dependent damage sensitivity and Pol II stalling following CPT. Similarly, they find in mammalian cells that sensitivity due to CTIP loss can be rescued by SETX expression and that RNA-DNA hybrids accumulate upon CTIP-loss or XPG-loss at laser-induced damage, based on binding of an mCherry-RNaseH sensor. Hybrids are also elevated in the absence of damage and these nucleases both at genes know to form R-loops and R-loop inducible sites. Finally, the authors show that CtIP and XPG reduce breaks formed by CPT using both comet assays and LM-PCR. Together, these studies lead the authors to propose that XPG and CtIP work in a redundant manner to process R-loops by generating a break at the R-loop site and allowing access of SETX to the hybrids for hybrid resolution.While this is an intriguing model and important function of CTIP to understand, I think that the model is somewhat premature at this point and additional results are needed to test the model and rule out other possibilities. Several experiments require additional controls as well. Of particular note, I don't think the authors have sufficiently established the proposed order of events which place CtIP and XPG upstream of SETX. Is CtIP or XPG needed for Sen1/SETX to bind the DNA at the sites of hybrid formation, tested using the RNaseH sensor and ChIP assays? This would be one prediction of the model if nuclease processing is needed to generate access for Sen1/SETX.As discussed in point #4 above, we agree that this is an important issue and we measured SEN1 recruitment as well as SETX recruitment in yeast and human cells, respectively. We do not find support for the idea that Sae2/CtIP recruits SEN1/SETX, although we do think it is possible that Sae2/CtIP creates an intermediate that is necessary for its function. We also consider other models, as discussed in the main text.Also, can the authors rule out that SETX and CtIP are not simply acting to reduce the same hybrid structures ? As it stands the authors need more data to substantiate their conclusions.9) and for DSBs occurring at sites of R-loops (10), we are also considering the possibility that an intermediate in this pathway is a DSB (see discussion in main text).In the case of CtIP, we are considering the fact that all of our evidence points toward a role for the nuclease activity in promoting removal of the R-loop, and the nuclease is clearly specific for a 5ʹ flap. We have not worked with XPG in vitro but the literature shows that it also is specific for a 5ʹ flap. If these activities are working at the site of an R-loop, there is no obvious way for either enzyme independently or both together to completely remove a hybrid. So, we have postulated in our working model that SETX is acting downstream of one or both of the nucleases, since it is known to be present at sites of hybrids and to play important roles in removing hybrids. Since there is substantial evidence for R-loops forming at DSB sites .We were able to work out the technical issues with DRIP in yeast and found that there are elevated R-loops at sites of high transcription and high Sae2 occupancy with DNA damage in S phase (see point # 3). We also find that the survival of Figure 3: The authors need to analyze several other genes that are not expressed or expressed at lower levels to show specificity of the effect of CtIP loss at highly expressed genes. Right now they examine two genes, PDC1 and NRD1 as negative controls (if I understand this correctly) and Pol II binding is actually elevated at the first site.We are not suggesting that Sae2 is at all highly-transcribed genes; there are many locations in the genome where transcription is high, yet we do not see Sae2 at these sites. NRD1 serves as a negative control here, where we see neither Sae2 nor accumulation of RNAPII.Figure 4: Does RNaseH overexpression rescue viability of CTIP deletion? Also, please comment why in Figure 4 hybrids increase with DRB treatment in WT cells?See point #2. The difference between wild-type untreated and wild-type with DRB is not statistically significant and are within the range we normally observe for wildtype cells.Figure 4, Figure 5 and Figure 6: Is the nuclease activity of XPG required for any of the observed effects? Testing this in at least one of the assays would be helpful.11). We would like to know if that role and the role we are examining in this study require the nuclease activity; however, these authors reported that it is not possible to reconstitute the function of XPG with respect to homologous recombination using a transgene or overexpression so at this point we have not attempted this. It is worth trying in the future though.We do not have the reagents to express recombinant XPG in human cells. It is interesting that XPG was reported to associate with BRCA1, which also binds directly to CtIP, and to participate in some way in homologous recombination . This is an important control.See point #1 above.Figure 5E/F: What is the rationale for both increases and decreases in transcription overlapping with site of hybrid formation? I don't understand the implications of the RNA-seq data. Also, Figure 5F is missing in the Figure, making these data difficult to evaluate.12). Figure 5F does not appear to be missing.RNA-DNA hybrids and DNA breaks can cause both increases as well as decreases in transcription  or accumulation in G2. It seems in WT cells hybrids are naturally higher in G2 and if breaks depend on S phase entry changes in cell cycle could affect the interpretation.We have measured cell cycle progression in CtIP-depleted, XPG-depleted, and double-depleted cells and find that is not significantly different from wild-type (data not shown). We are not removing 100% of either of these factors, which may explain why we do not observe drastic changes in cell growth or cell cycle progression .Figure 6: Why are there no more breaks upon addition of CPT? Shouldn't the CPT itself lead to break formation even without the action of XPG and CtIP? Also, alkaline comet is not specific for SSB detection – these could be DSBs. I suggest the authors use caution in the way they present these data.There are significantly more ssDNA breaks observed with CPT treatment in wild-type cells. We are showing the single-strand breaks here because we see an obvious change with DNA damage treatment and with the status of CtIP and XPG, whereas the neutral comet assay shows comparatively few breaks under the conditions used in these experiments. It is true that DSBs would also be evident in an alkaline comet assay but if this was the origin of the signal, they would be evident also in the neutral assay.The idea that the R-loops are too small to be detected when both XPG and CtIP are lost is not well supported. Isn't it possible that there is no R-loop under these conditions and that RNAPII has simply stalled and is the toxic lesion?Yes, this is possible. See discussion of this issue in the main text.Can RNaseH expression rescue sensitivity due to loss of both XPG and CtIP? That is one test of this idea. Or can evidence for a smaller R-loops be obtained using bisulfite sequencing. Minimally I think other models should be considered.We have not done RNaseH rescue of XPG+CtIP depletion. We discuss other scenarios in the main text.[Editors' note: the author responses to the re-review follow.]Summary:Makharashvili et al., report a novel function for Sae2/CtIP in both yeast and human cells, and propose that Sae2/CtIP, known to promote resection during DSB repair, also contributes in R-loop (DNA-RNA hybrids) dissolution, in conjunction with XPG. In this revised version, the authors have addressed to a reasonable extent my main points. More specifically they are now consistent throughout the manuscript, and report Sae2 binding and R-loop accumulation in Sae2 deleted strain at the same loci. They have also now changed their discussion to take more hypotheses into account. They have also largely controlled their data by performing RNAseH treatment to validate their results obtained with S9.6 antibody. This manuscript is a significantly improved version of the previous one. Results are now clearer and convincing. However, the key conclusion of their model that CtIP cleaves the loop needs further support and clarification before acceptance.Essential revisions:The results on which the model proposed rely on CPT-treated cells  (CPT creates ssDNA breaks with Top1-cc) or cells upon laser-induced breaks . So, the experimental conditions used implies to start with a genotoxic-induced break. It is still therefore unclear why CtIP would be required for an additional break. Indeed, authors were not able to show the proposed activity of CtIP in vitro. Authors would need to show the action of CtIP in untreated cells , with no break, to support their model that demands that CtIP cleaves the loop. The known action of the Sae2/CtIP nuclease activity on DNA resection as a requirement to remove the RNA-DNA hybrid accumulated at a break would make much more sense and fit better with the combined effect with XPG. Therefore, either authors provide new data with untreated cells to support their model as such or alternatively include in their model the breaks caused by CPT/laser irradiation to explain the role of CtIP under such conditions.In the current version of the manuscript, we show that CtIP depletion in untreated cells generates accumulation of R-loops . Figure 5B specifically shows the effect of the nuclease-deficient mutant in untreated cells. Also, our quantification of ssDNA breaks at the RPL13A locus in Figure 8D is in untreated cells.The reviewers request that existing (or exogenously induced) DNA breaks be incorporated into the model, since some of our assays utilize agents that induce breaks and under these conditions, we observe an effect of Sae2/CtIP that relates to transcription-induced DNA damage.We have incorporated a block with an adjacent nick into a revised version of the model in Figure 7. This version also shows the nucleic acids in a manner that is closer to the most recent structures of the elongating RNA polymerase holoenzyme .References:H. Ehara et al., Structure of the complete elongation complex of RNA polymerase II with basal factors. Science. 357, 921–924 (2017).


The major objective of this work is to study the hardness data at the domain of ferrite and Austenite phases. Nanoindentation and microhardness study has been conducted on austenite and ferrite present in the microstructure of hot rolled and heat treated duplex stainless steel (2205 DSS). Furthermore, Optical microscopy and field emission scanning electron microscope (FE-SEM) were used to identify the microstructural distribution and phases present. Austenite reveals higher nanohardness data value than ferrite, as oppose to ferrite average elastic modulus which is higher than that of austenite. Also, higher value of microhardness data was observed for austenite in comparison with the ferrite at different load application. The chemical composition data analysis of the as received DSS (2205) is shown in 22.1The material investigated in this research was obtained from Columbus stainless (pty) Ltd. as 6mm 2.2The nanoindentation was carried out on an ultra-nanoindenter (UNHT), manufactured in Switzerland, equipped with a three-sided pyramid, berkovich diamond indenter. All the indentation tests followed ISO 14577. Before the indentation tests, the contact area was calibrated by an indirect method to maintain accuracy. The total indentation time is 40s which was divided using load control function with a 20s loading time, 5s holding time and 15s unloading time. Also, varying load from 30mN to 50mN, and the grid of indents was spaced 2 According to Tao et al. From Kicks Law Showing that the load is directly proportional to the square value of indentation displacement. WhereThe initial unloading curveThe indentation hardness and elastic modulus was obtained from Oliver -Pharr analysis 2.3The Vickers microhardness (HV) was measured by Vickers microhardness tester (FUTURE-TECH FM 800) at a load (P) 20 gf (1.0 N)- 200gf and a dwell time of 10 s at room temperature, with five repeat tests to ensure data reliability.The Vickers microhardness test was conducted on both the ferrite and austenite phases. Meanwhile, the Vickers microhardness experimental data, indicated that there was no significant difference between the hardness value of the austenite phase and the ferrite phase except for the hardness test carried out at 50g load giving an average hardness values of 343 HV and 311 HV for ferrite. However, we can conclude and assume that, the factors responsible for high Nano hardness can also be the same factors for austenite high micro hardness.


Background: A prospective cohort study was conducted to follow-up on 104 participants on their changes of social, psychological and physical health as exposed to the hikikomori lifestyle. Methods: Participants were interviewed at baseline, 6 months and 12 months by administering a set of questionnaires and anthropometric measurements. Results: All three health domains of hikikomori were significantly improved over the follow-up period as evidenced by: (1) increased social network scores from 2.79 ± 1.80 to 3.09 ± 1.87, (2) decreased perceived stress scores from 21.18 ± 5.87 to 20.11 ± 5.79, and (3) reduced blood pressure levels from 118/75 to 115/71 and waist-to-hip ratios. Almost half of the participants have recovered from hikikomori by returning to the workforce in society; however, the health improvements were dominant in those that remained as hikikomori and were associated with the gradual swapping of exercise practices from light to moderate level strength. Conclusions: With intended exposure to social worker engagement, physical assessments of the cohort study triggered the social workers to encourage participants to do more exercises, which in turn enhanced their awareness of health modification towards a better health. Engagement of social workers could be considered as part of the intended exposure for all participants, which suggested social work intervention was effective in helping hikikomori recovery. The Japanese term “Hikikomori” (translated as 隱蔽青年 in Chinese) describes both the condition and identity of a severe form of social withdrawal. The universal definition adopts any individual who without a clear or legitimate purpose confined him/herself at home for more than 6 months, avoiding face-to-face contact with others except family or a close person, and having a ‘Status Zero’—meaning not participating in education, training, or work ,2. By usThis is a prospective cohort study designed to follow-up 104 hikikomori cases at 6 months (time point 2) and 12 months (time point 3) regarding the changes in their social, psychological and physical health while exposed to the sedentary lifestyle as reported in our previous publication [Participants were approached by their case social workers to make an appointment for interview  about one month before the study follow-up. On each interview day, the data collector (a trained nursing student) followed the respective social worker on their home visits to the potential participants, and conducted the measurements in the absence of the social worker immediately after their consultation. The Psychotic Screening Module of the Structured Clinical Interview for DSM Disorders Axis I (SCID-I) was used to screen the participants at each time point to exclude any psychotic and associated symptoms. Participants clinically diagnosed with mood disorders at follow-ups were also excluded. Exclusion of such participants with psychosis and clinical mood disorders was because they might not be able to complete the comprehensive set of questionnaires. However, none of the participants had psychotic ineligibility, therefore this exclusion criterion had no impact in this study. And then, the participants were proceeded with the same procedure of physical measurements and completing a set of self-administered questionnaires as performed at baseline. The interview lasted around 45–60 min. A cash voucher was given to each participant at the end of each interview as a token of appreciation. The data collectors were well trained, particularly a 20-h training was provided for the semi-structured SCID-I with the use of the instrument training kit as specified by the developer. Inter-rater reliability was assessed prior to the data collection until satisfactory agreement was achieved among all data collectors .The instrument consisted of the same set of questionnaires used in the previous publication, where the scales’ reliability scores and interpretation methods have also been described alongside with the baseline results . In brieAfter the baseline measurement (time point 1), the PSS-14 scores were compared and found to be consistently compatible after removing the 4 items . Concerning the long instrument to be administered by the participants, the PSS-10 was adopted since the 6 month follow-up, and hence the instrument length was shortened from a total of 156 items to 152 items for easier administration.Data collected in this study were analyzed using IBM SPSS Statistics 22.0 . Frequency and percentage were computed for each of the binary or categorical variables, whereas mean and standard deviation (SD) were computed for continuous variables. Together with the anthropometric variables measured in this study, the composite scores were computed for all subscales of the instrument according to the subscale scoring schemes, and expressed as mean and SD. Missing data was replaced by the last observation carry forward method. Those continuous variables were compared across the three time points using the Repeated Measures Analysis of Variance NOVA (RMANOVA) was used to examine the changes over the three time points, whereas the F values and imputed p-values were reported. Additionally, Generalized Estimating Equation (GEE) was performed as an alternative method for RMANOVA to see whether the trend is significant or not.From March 2017 to June 2018, the cohort of 104 hikikomori that reported with the baseline health profile (time point 1) [p < 0.001) by 5.0% in time point 2 and 10.8% in time point 3 as compared with the baseline  and any of its four subscales. Regarding the mental health, the perceived stress levels (PSS-10 scores) of participants were significantly reduced across the three time points . As compp < 0.001. Similar reducing trend was also observed for the SBP, making the reduction of SBP/DBP of 118/75 (SD = 16/10) at baseline to 117/73 (SD = 15/9) at time point 2 and 115/71 (SD = 13/9) at time point 3 (p = 0.024) was shown in the waist-to-hip ratios of participants throughout the follow-ups, which was consistent with the decreasing trend of the waist circumference  socially with increasing social networks, both online and offline, (2) mentally with decreasing perceived stress levels, and (3) physically with reduced blood pressure levels as well as waist-to-hip ratios.As reported in our previous study, a number of health manifestations associated with hikikomori, such as hypertension and obesity could be at least partially contributed by their sedentary lifestyle . This caCould such health improvements be explained by any changes of the living lifestyle? In the current study, no significant lifestyle changes were observed over the study period, except for the upward trend of practicing moderate-intensity exercises. The joint guidelines of World Health Organization and International Society of Hypertension ,32 suggeHowever, what caused the hikikomori to exercise more intensely? Current findings suggested that participants remained as hikikomori were shown to have better health improvements than those who recovered. Whereas the gradually swapping of exercise practices from light to moderate level strength (increase of exercise intensity) was only observed amongst those remained as hikikomori, which further suggested the participants were readily to adopt a healthier lifestyle even though without recovery. As mentioned in a previous publication , empiricGiven that this study adopted a prospective cohort design, no structural intervention was provided to the participants. However, all participants of this prospective cohort study were recruited through the case social workers who had been providing them ongoing counseling and psychosocial social support. Those case social workers had the primary goal to find jobs or study opportunities for their clients and get them ready to return back to the society in a gradual and voluntarily manner. In this sense, the engagement of social workers was an intended exposure for all participants of this study. The fact indicated that such social work intervention was effective, since almost half of the participants have returned to the workforce or study institutions within a year time. The role of social workers seemed to be crucial for both preferred outcomes of hikikomori recovery and health promotion during the hikikomori stage. Therefore, a qualitative study is planned to understand the views of the social workers according to their experience in dealing with clients receiving the contemporary health assessment. Furthermore, the current study and Hong Kong social workers’ approach could also be inspiring the practice of other places. According to the recent Japanese Cabinet survey , the hikThere are, however, several limitations in this study. Because of the hidden nature of the target participants who are one of the hardest groups for engagement and rapport building following their protracted period of seclusion at home, subject recruitment is considered as the most difficult part of the study. It caused the small sample size and high attrition rate as a major limitation. Although the sample size was sufficiently enough to achieve statistical significance when certain measured variables were compared, the high attrition rate due to loss of follow-up would contribute to selection bias that threatening to the internal validity . ParticuThe hikikomori lifestyle which is largely sedentary in nature that could be a risk behavior, but a longitudinal study has observed improvements in all three domains of the health profile, namely social networks, perceived stress, and blood pressure levels . The reduction of blood pressure levels and prevalence were consistent with the reduction of waist-to-hip ratios as well as the increase of moderate-intensity exercise over the follow-up period. Whilst the reduction of perceived stress was more specifically associated with the participants recovering from hikikomori, physical assessments followed by encouragement from social workers to do more exercises might enhance awareness of hikikomori youths in health modification towards a better health. There is implication for promoting inter-disciplinary collaboration across healthcare and social care sectors for conducting further empirical studies and delivering engagement interventions at a pace that secluded and marginalized hikikomori find comfortable.