Cohesin is involved in transcriptional repression of stage-specific genes in the human malaria parasite

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   Review Commons

   Feb 28, 2023

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   Reply to the reviewers

   We thank all the reviewers for having raised constructive criticism to fortify the main message and improve the clarity of the manuscript. We appreciate that all reviewers found that our work addresses an important topic and is of interest to a broad audience. We believe that we have thoroughly addressed the concerns of the reviewers, especially with regard to 1) performing another SMC3 chromatin immunoprecipitation and sequencing (ChIP-seq) replicate and control, 2) including a later time point for the transcriptional data, and 3) performing additional characterization of the growth phenotype of the SMC3 conditional knockdown.

   Reviewer #1

   (Evidence, …

   Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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   Reply to the reviewers

   We thank all the reviewers for having raised constructive criticism to fortify the main message and improve the clarity of the manuscript. We appreciate that all reviewers found that our work addresses an important topic and is of interest to a broad audience. We believe that we have thoroughly addressed the concerns of the reviewers, especially with regard to 1) performing another SMC3 chromatin immunoprecipitation and sequencing (ChIP-seq) replicate and control, 2) including a later time point for the transcriptional data, and 3) performing additional characterization of the growth phenotype of the SMC3 conditional knockdown.

   Reviewer #1

   (Evidence, reproducibility and clarity (Required)):*

   Summary The present work by Rosa et al., provides convincing data about the presence and functional relevance of the cohesin complex in Plasmodium falciparum blood stages. In accordance with other organisms, the composition of the cohesin complex containing SMC1, SMC3 RAD21 and putatively STAG could be confirmed via pulldown and mass spectrometry. Basic characterization of endogenous tagged SMC3 demonstrated the expression and nuclear localization during IDC, as well as the relatively stable accumulation at centromeric regions, consistent with the known cohesin function in chromatid separation. Furthermore, dynamic and stage-dependent binding to intergenic regions observed in ChIPseq and major transcriptome aberrations upon knockdown of SMC3 (__Response: __As we regularly perform ChIP-seq experiments in the lab, we have generated multiple negative control datasets. In our opinion, the most stringent negative control for an HA-tagged protein is performing ChIP with an HA antibody in a WT strain. We have recently published an in-depth analysis of this (and other) negative ChIP-seq controls (Baumgarten & Bryant, 2022, https://doi.org/10.12688/openreseurope.14836.2). We show in this publication that non-specific ChIP-seq experiments (such as negative controls) result in an over-representation of HP1-heterochromatinized regions due to differences in sonication efficiency of heterochromatin and technical challenges with mapping regions with high levels of homology. In the anti-HA in WT ChIP negative control (performed at 12hpi), we do not see any enrichment at centromeric regions, but rather at heterochromatinized regions where clonally variant gene families are located. We performed peak calling analysis and found no significant overlap between the negative control ChIP-seq and the SMC3-3HA ChIP-seq data at 12hpi.

   In addition, we have now performed a second biological replicate of the SMC3-3HA ChIP-seq with a different clone at all time points. We compared this data to that from the original clone and found significant overlap of the peaks called (see what is now Table 4 and Supp. Fig. 3A). We generated a stringent list of peaks that were shared between both clones at each time point and repeated all downstream analyses (see what are now Tables 5-8). We found that our conclusions were largely unchanged. Text describing these experiments and analyses have been added throughout the results section.

   • Proposed mechanism of repressive effect of SMC3 early in IDC on genes, that get de-repressed in late stages: To claim this mode of function, it would be necessary to include a KD on late stage parasites. If there is an early repressive role of SMC3, upregulated genes should not be affected by late SMC3-KD. __Response: __To be clear, we are most interested in the transcriptional role of SMC3 during interphase, where results are not confounded by its potential role in mitosis. However, we did collect a 36hpi time point in the SMC3-3HA-glmS and WT strain, with and without glucosamine. We have added this last time point and the WT data from the other two time points to the manuscript (see Tables 11-13). Unfortunately, and for reasons unknown, the WT replicates treated with glucosamine showed a significantly advanced “transcriptional age” compared to the other replicates at 36hpi (see what is now Supp. Fig. 5B). Thus, we did not feel comfortable performing the RNA-seq analysis as we did with the other two time points (i.e. subtracting up- and down-regulated genes from the WT control from the SMC3-3HA-glmS data sets). We have added this information to the results section (Lines 256 and 261). As the WT parasites treated with glucosamine were approximately 8 hours in advance of the untreated WT parasites for the 36hpi time point, any up- and down-regulated genes might have been due to differences in the cell cycle rather than due to glucosamine treatment. The glmS system of inducible knockdown is widely used in P. falciparum; however, to our knowledge, no lab has investigated whether glucosamine treatment affects transcription in wildtype cells over the course of the IDC. Thus, for accurate phenotypic characterization of any protein with this system with regard to transcriptomics, we thought it was important to provide an RNA-seq dataset to define the cohort of genes affected by glucosamine treatment in WT parasites. We hope that our study will demonstrate the importance of using stringent controls when using inducible knockdown systems.

   To address the question of whether genes that are upregulated upon depletion of SMC3 at early stages are affected at the 36hpi time point, we performed differential expression analysis of the SMC3-3HA-glmS parasites with and without glucosamine at 36hpi (we have added this data in Table 11). Again, significantly up- and down-regulated genes were not filtered using the WT dataset. With this analysis, we see only three genes from the list of invasion-related genes (Hu et al., 2010) that are up-regulated, but none of them have a significant q-value (Tab 5 of Table 18). Thus, depletion of SMC3 in late stage parasites does not lead to up-regulation of the same genes that are upregulated at 12 and 24hpi. We have added this information to the text (Line 273).

   Furthermore, the hypothesized repressive effect of SMC3 does not explain the numerous genes downregulated in KD.

   __Response: __As we state on line 350, we do not observe enrichment of SMC3 at downregulated genes, suggesting an indirect or secondary effect of SMC3 KD on these genes.

   • Due to the fact, that the KD was induced at the exact same timepoint and analysed 12h and 24h after induction it is possible that identified, differentially expressed genes at 24h are not directly regulated by SMC3, but rather due to a general deregulation of gene expression. Did the authors attempt to analyse gene expression upon induction at ring, trophozoite and schizont stage? __Response: As we state on line 230, in order to achieve SMC3 KD at the protein level, we had to treat the parasite with glucosamine for two cell cycles (approximately 96 hours). After two cell cycles of glucosamine treatment, the parasites were tightly synchronized and sampled 12 and 24 hours later. Thus, SMC3 KD takes place over the course of multiple days, but parasites are collected after stringent synchronization. Giemsa staining and bioinformatic analysis (line 250) of the RNA-seq data from parasites (with or without glucosamine) harvested at 12 and 24 hpi show that these parasites were synchronous and that there were no gross differences in genome-wide transcript levels. It is certainly possible that differentially expressed genes at 12 or 24hpi are not directly regulated by SMC3, and this is precisely why we perform ChIP-seq of SMC3: to provide evidence of direct involvement via binding, as stated on line 281. __

   • *Based on rapid parasite growth, the authors hypothesize a higher invasion rate due to upregulation of invasion genes. This hypothesis is not supported by quantitative invasion assays or quantification of invasion factors on the protein level. An alternative explanation could be a shorter cell cycle (__Response: __We have repeated the growth curve analysis with additional clones and no longer observe a growth phenotype in the SMC3 knockdown condition. We have added images of Giemsa-stained parasites from the knockdown time course we performed to what is now Supp. Fig. 5A. We see no obvious differences in cell morphology caused by glucosamine treatment in the WT or SMC3-3HA-glmS parasites.

   • Correlation of SMC3-occupancy/ATAC/expression profile of the exemplary genes rap2 and gap45 (Figure 4C,D,E): is this representative for all upregulated genes? __Response: __SMC3 occupancy shown at rap2 and gap45 is representative for all upregulated genes (see Fig. 4A and B). It is difficult to provide a general representation of the average expression profiles of all up-regulated genes over the course of the IDC, but Fig. 3E shows that the vast majority of up-regulated genes normally reach their peak expression in late stage parasites. With regard to ATAC-seq profiles, we have performed a metagene analysis of chromatin accessibility (data taken from (Toenhake et al., 2018)) at all up-regulated genes at time points that closely correspond to the time points used in our study: 15, 25, and 35, and 40 hpi (new Fig. 4C). This metagene analysis confirms what we observe at individual genes: increasing chromatin accessibility over the course of the IDC at these genes’ promoters. While metagene analyses offer important information, we always try to show the raw data (as in new Figs. 4D-F) from individual examples as proof of principle.

   • Given that SMC3 appears to be not essential for parasite growth, the authors could generate a null mutant for SMC3, which might allow for easier analysis of differences in gene regulation, cell cycle progression and/or invasion efficiency. __Response: __As we explain on line 327, very little cohesin is required for normal growth and/or mitosis in our study and two studies in S. cerevisiae and D. melanogaster. However, SMC3 is essential in S. cerevisiae. We were unable to knock out SMC3, and a recent mutagenesis study suggests that SMC3 and SMC1 are essential to the parasite during the intraerythrocytic developmental cycle (Zhang et al. Science, 2018). This is why we chose an inducible knockdown system.

   *Reviewer #1 (Significance (Required)):

   Own opinion The authors provide a basic characterization of the cohesin component SMC3 using NGS methods to investigate chromatin binding sites and its potential influence on gene expression. *

   __Response: __We respectfully disagree that our study offers only a basic characterization of SMC3. We combine IFA, mass spectrometry, and both ChIP-seq and RNA-seq of SMC3 across the entire intraerythrocytic developmental cycle to provide the most detailed and comprehensive functional analysis of SMC3 in P. falciparum to date.

   The localisation of SMC3 at centromers as described previously (Batugedara 2020) was confirmed. However, the dynamic binding to other regions in the genome, potentially mediated by other proteins, could not be resolved unequivocal with only one replicate of ChIPseq per time point.

   __Response: __With regard to the replicates for ChIP-seq, please see our response to this same point above.

   Similarly, the RNAseq data demonstrate the relevance of SMC3 for gene expression, but no clear picture of a regulatory mechanism can be drawn at his point. Lacking information about the mode of binding as well as the setup of transcriptome analysis (only two time-shifted sampling points after simultaneous glmS treatment for 96h resulting in incomplete knockdown) cannot definitely elucidate, if SMC3/cohesin is a chromatin factor that affects transcription of genes in general or a specific repressor of stage-specific genes. __Response: __We agree that we have not established a regulatory mechanism for how SMC3 achieves binding specificity. However, the combination of inducible knockdown (as SMC3 is essential to the cell cycle) and differential expression analysis with ChIP-seq from the same time points across the intraerythrocytic developmental cycle is the most stringent and standard approach in the field of epigenetics for determining the direct role of a chromatin-associated protein in gene expression. We provide a detailed explanation of how the transcriptome analysis was set up in the Results (lines 229-234) and Materials and Methods (lines 715-719) section. With regard to our sampling points being “time-shifted,” we provide bioinformatic analysis (line 246-251, what is now Supp. Fig. 5B) of the RNA-seq data from untreated and glucosamine-treated parasites showing highly similar “ages” with regard to progression through the intraerythrocytic developmental cycle. While we of course also monitor progression through the cell cycle with Giemsa staining (Supp. Fig. 5A), this bioinformatic analysis is the most stringent method of determining specific times in the cell cycle.

   *The work will be interesting to a general audience, interested in gene regulation and chromatin remodelling

   The reviewers are experts in Plasmodium cell biology and epigenetic regulation.*

   *Reviewer #2 *

   (Evidence, reproducibility and clarity (Required)):

   *Rosa et al, Review Commons The manuscript by Rosa et al. addresses the function of the cohesion subunit Smc3 in gene regulation during the asexual life cycle of P. falciparum. Cohesin is a conserved protein complex involved in sister chromatin cohesion during mitosis and meiosis in eukaryotic cells. Cohesin also modulates transcription and DNA repair by mediating long range DNA interactions and regulating higher order chromatin structure in mammals and yeast. In P. falciparum, the Cohesin complex remains largely uncharacterized. In this manuscript, the authors present mass spectrometry data from co-IPs showing that Smc3 interacts with Smc1 and a putative Rad21 orthologue (Pf3D7_1440100, consistent with published data from Batugedara et al and Hilliers et al), as well as a putative STAG domain protein orthologue (PF3D7_1456500). Smc3 protein appears to be most abundant in schizonts, but ChIPseq indicates predominant enrichment of Smc3 in centromers in ring and trophozoite stages. In addition, Smc3 dynamically binds with low abundance to other loci across the genome; however, the enrichment is rather marginal and only a single replicate was conducted for each time point making the data interpretation difficult. Conditional knock-down using a GlmS ribozyme approach indicates that parasites with reduced levels of Smc3 have a mild growth advantage, which is only evident after five asexual replication cycles and which the authors attribute to the transcriptional upregulation of invasion-linked genes following Smc3 KD. Indeed, Smc3 seems to be more enriched upstream of genes that are upregulated after Smc3 KD in rings than in downregulated genes, indicating that Smc3/cohesin may have a function in supressing transcription of these schizont specific genes until they are needed. The manuscript is concise and very well written, however it suffers from the lack of experimental replicates for ChIP experiments and a better characterization of the phenotype of conditional KD parasites.

   Major comments • In the mass spectrometry analysis, many seemingly irrelevant proteins are identified at similar abundance to the putative rad21 and ssc3 orthologues, and therefore the association with the cohesion complex seems to be based mostly on analogy to other species rather than statistical significance. Hence, it would be really nice to see a validation of the novel STAG domain and Rad21 proteins, for example by Co-IP using double transgenic parasites.

   __Response: __While our IP-MS data did not yield high numbers of peptides, the top most enriched proteins were SMC3 and SMC1. As we state on line 157, two previous studies have already shown a robust interaction between SMC1, SMC3, and RAD21 in Plasmodium, supporting the existence of a conserved cohesin complex. While the identification of the STAG domain-containing protein is interesting, the purpose of our IP-MS was less about redefining the cohesin complex in P. falciparum and more about confirming that the epitope-tagged SMC3 we generated was incorporated correctly into the cohesin complex and was specifically immunoprecipitated by the antibody we later use for western blot, immunofluorescence, and ChIP-seq analyses. However, to validate the results of ours and others’ mass spectrometry results, we generated two new parasite strains – SMC1-3HA-dd and STAG-3HA-dd – and an antibody against SMC3 (see what is now Supp. Fig. 1). We performed co-IP and western blot analysis with these strains and show an interaction between SMC1 and SMC3 and STAG and SMC3 (see what is now Supp. Fig. 2). This information has been added to the manuscript on lines 162-167.

   • *The ChIPseq analysis presented here is based on single replicates for each of the three time points. The significance cutoffs for the peaks are rather high (q __Response: __In our experience, a significance cutoff of FDR As we regularly perform ChIP-seq experiments in the lab, we have generated multiple negative control datasets. In our opinion, the most stringent negative control for an HA-tagged protein is performing ChIP with an HA antibody in a WT strain. We have recently published an in-depth analysis of this (and other) negative ChIP-seq controls (Baumgarten & Bryant, 2022, https://doi.org/10.12688/openreseurope.14836.2). We show in this publication that non-specific ChIP-seq experiments (such as negative controls) result in an over-representation of HP1-heterochromatinized regions due to differences in sonication efficiency of heterochromatin and technical challenges with mapping regions with high levels of homology. In the anti-HA in WT ChIP negative control (performed at 12hpi), we do not see any enrichment at centromeric regions, but rather at heterochromatinized regions where clonally variant gene families are located. We performed peak calling analysis and found no significant overlap between the negative control ChIP-seq and the SMC3-3HA ChIP-seq data at 12hpi.

   In addition, we have now performed a second biological replicate of the SMC3-3HA ChIP-seq with a different clone at all time points. We compared this data to that from the original clone and found significant overlap of the peaks called (see what is now Table 4 and Supp. Fig. 3A). We generated a stringent list of peaks that were shared between both clones at each time point and repeated all downstream analyses (see what are now Tables 5-8). We found that our conclusions were largely unchanged. Text describing these experiments and analyses have been added throughout the results section.

   The SMC3 ChIP from Batugedara et al., 2020 was performed with an in-house generated antibody (not a commercially available, widely validated antibody as we use) at a single time point in the IDC: trophozoites. Batugedara et al. performed one replicate and did not have an input sample for normalization. Rather, it seems that they incubated beads, which were not bound by antibody or IgG, with their chromatin and used any sequenced reads from this beads sample to subtract from their SMC3 ChIP signal as means of normalization. According to ENCODE ChIP-seq standards, this is not a standard nor stringent way of performing ChIP-seq and the subsequent analysis. Because they did not generate a dataset for their ChIP input, it is not possible to call peaks as we do in our study and compare those peaks with ours.

   • The authors argue that during schizogony, cohesin may no longer be required at centromers, explaining the low ChIPsignal at this stage (Line 301). However, during schizogony parasites undergo repeated rounds of DNA replication (S-phase) and mitosis (M-phase) to generate multinucleated parasites; and concentrated spots of Smc3 are observed in each nucleus in schizonts by IFA. In turn, the strong presence of Smc3 at centromers in ring stage parasites is surprising, particularly since the Western Blot in Figure 1D shows most expression of Smc3 in schizonts and least in rings; and Smc3 is undetectable in rings by IFA. Yet, the ChIP signal shows very strong enrichment at centromers, long before S phase produces sister chromatids. What could be the reason for this discrepancy? Again, ChIP replicates and controls would be helpful in distinguishing technical problems with the ChIP from biologically relevant differences. __Response: __We discuss in lines 337-342 not that cohesin is no longer required at centromeres during schizogony, but that its removal from centromeres may be required specifically for separation of sister chromatids, as is seen in other eukaryotes. We also discuss that the unique asynchronous mitosis in Plasmodium may lead to a mixed population of parasites at the time point sampled where there may be some centromeres with SMC3 present and some where it is absent to promote sister chromatid separation. Even though SMC3 may be evicted from centromeres to promote sister chromatid separation, it is likely re-loaded onto centromeres once this process is complete. This is most likely why we see foci of SMC3 in each nucleus of mature schizonts by IFA. With regard to the discrepancy between SMC3 levels in rings seen in total nuclear extracts (by western blot) and at centromeres (by ChIP-seq): the total level of a protein in the nucleus does not necessarily dictate the genome-wide binding pattern or the level of enrichment of that protein at specific loci in the genome. Moreover, if one molecule of SMC3 binds to each centromere, 14 molecules would be needed in a ring stage parasite while over 500 would be needed in a schizont (assuming that there are ~36 merozoites present). SMC3 binds to centromeres in interphase cells in other eukaryotes as well, and we speculate that this binding may play a role in the nuclear organization of centromeres, as we discuss starting on line 333.

   • It is surprising that a conserved protein like Smc3 shows such a subtle phenotype, given that it is predicted to be essential and its orthologues have a function in mitosis. Generally, only limited data are presented to characterize the Smc3 KD parasites, and more detail should be included. For example validation of the parasite line using a PCR screen for integration and absence of wt, parasite morphology after KD, and/or analysis of the KD parasites for cell cycle status. __Response: __First, we have repeated our growth curve analysis several times and with more clones and have concluded that there is not a significant growth phenotype in SMC3 KD parasites (see what is now Supp. Fig. 4B). As we discuss on line 342, very little intact cohesin complex seems to be required for normal growth and mitosis in S. cerevisiae and D. melanogaster, which is probably why we do not see an obvious growth or morphological phenotype. Because we could not generate SMC3 knockout parasites, there may be just enough SMC3 left to perform its vital function in our KD strain. We have added PCR data to demonstrate integration of the 3HA tag- and glmS ribozyme-encoding sequence in the clonal strains we are using for all experiments (see what is now Supp. Fig. 1A). Sanger sequencing was performed on these PCR products to confirm correct sequences. We also added images of Giemsa-stained parasites in untreated and glucosamine-treated parasites at all time points to demonstrate a lack of an obvious morphological phenotype in SMC3 KD parasites (see what is now Supp. Fig. 5A).

   • Synchronization was performed at the beginning of the growth time course, which would be expected to result in a stepwise increase in parasitemia every 48 hours; however, the parasitemia according to Fig. 4F rises steadily, which would indicate that the parasites are actually not very synchronous. __Response: __We did indeed tightly synchronize these parasites and hope that the stepwise increase in parasitemia is seen better in our new growth curve analysis (see what is now Supp. Fig. 4B).

   • The question of whether Smc3 causes a shorter parasite life cycle (quicker progression) or more invasion is important and could be experimentally addressed by purifying synchronous schizont stage parasites and determining their invasion rates as well as morphological examination of the Giemsa smears over the time course. __Response: __We have repeated our growth curve analysis several times and with more clones and have concluded that there is not a significant growth phenotype in SMC3 KD parasites (see what is now Supp. Fig. 4B).

   • Please also compare Smc3 transcriptional levels in transgenic parasites to those in wt parasites to rule out that the genetic modification has lead to artificial upregulation of Smc3 transcription. __Response: __We have added this data to what is now Supp. Fig. 4C, showing that there is no significant difference in SMC3 transcript levels between WT and SMC3-3HA-glmS strains. We have added this information to the text of the manuscript (Line 243). As we also generated an SMC3 antibody, we could demonstrate that there is no appreciable difference in SMC3 protein levels between WT and SMC3-3HA-glmS strains (see what is now Supp. Fig. 1D).

   • According to Figure S2, even more genes were deregulated at the 12 hpi time point in the WT parasites than in Smc3 parasites, and even to a much higher extent. What "transcriptional age" did the WT control parasites have at each time point? __Response: __We have now included the transcriptional age of all strains, replicates, and treatments in what is now Supp. Fig. 5B. At the 12 hpi time point in particular, regardless of glucosamine treatment, the SMC3-3HA-glmS and WT parasites were highly synchronous. The only large discrepancy we see in transcriptional age is between untreated and glucosamine-treated WT parasites at 36 hpi, which is why we did not include this time point in our transcriptional analysis. We were also surprised by the number of genes that were de-regulated with simple glucosamine treatment. The glmS system of inducible knockdown is widely used in P. falciparum; however, to our knowledge, no lab has investigated whether glucosamine treatment affects transcription in wildtype cells over the course of the IDC. Thus, for accurate phenotypic characterization of any protein with this system with regard to transcriptomics, we thought it was important to provide an RNA-seq dataset to define the cohort of genes affected by glucosamine treatment in WT parasites. We hope that our study will demonstrate the importance of using stringent controls when using inducible knockdown systems.

   • A negative correlation with transcription is well established in S. cerevisiae, particularly at inducible genes. How does Smc3 enrichment generally look like for genes that show maximal expression at each of the time point? __Response: __We have performed a metagene analysis of SMC3 enrichment at all genes at each respective time point, which we divided into quartiles of expression based on their FPKM values in the RNA-seq data from the corresponding time point in untreated SMC3-3HA-glmS parasites. This quartile analysis considers all genes, including genes that are not transcribed at all and regardless of whether a gene has a significant SMC3 peak or is differentially expressed upon SMC3 knockdown. At the 12 hpi time point, we do see an inverse correlation between SMC3 enrichment and gene transcription level, but this enrichment is most pronounced across genes bodies. We see the highest SMC3 enrichment at genes in the 4th (lowest) quartile category. For the other two time points, we do not see any obvious pattern of SMC3 enrichment with regard to transcriptional status.

   • Line 590: according to the methods, a 36 hpi KD time point was also harvested. Why are the data not shown/analysed? __Response: __To be clear, we are most interested in the transcriptional role of SMC3 during interphase, where results are not confounded by its potential role in mitosis. However, we did collect a 36hpi time point in the SMC3-3HA-glmS and WT strain, with and without glucosamine. We have added this last time point and the WT data from the other two time points to the manuscript (see Tables 11-13). Unfortunately, and for reasons unknown, the WT replicates treated with glucosamine showed a significantly advanced “transcriptional age” compared to the other replicates at 36hpi (see what is now Supp. Fig. 5B). Thus, we did not feel comfortable performing the RNA-seq analysis as we did with the other two time points (i.e. subtracting up- and down-regulated genes from the WT control from the SMC3-3HA-glmS data sets). We have added this information to the results section (Lines 256 and 261). As the WT parasites treated with glucosamine were approximately 8 hours in advance of the untreated WT parasites for the 36hpi time point, any up- and down-regulated genes might have been due to differences in the cell cycle rather than due to glucosamine treatment. The glmS system of inducible knockdown is widely used in P. falciparum; however, to our knowledge, no lab has investigated whether glucosamine treatment affects transcription in wildtype cells over the course of the IDC. Thus, for accurate phenotypic characterization of any protein with this system with regard to transcriptomics, we thought it was important to provide an RNA-seq dataset to define the cohort of genes affected by glucosamine treatment in WT parasites. We hope that our study will demonstrate the importance of using stringent controls when using inducible knockdown systems.

   Minor Comments • Line 103/104: the hinge domain and ATPase head domain are mentioned, please annotate these in Figure 1A.

   __Response: __We have annotated the hinge and ATPase domains.

   • Figure 1D: the kDa scale is missing from the H3 WB. __Response: __We have added a kDa scale.

   • What is the scale indicated by different colors in Fig. 2A? __Response: __The different colors (blue, coral, and green) only represent the 12, 24, and 36hpi time points, respectively. This color scheme is used throughout the manuscript. If the reviewer is referring to the color gradation within each circos plot, this does not indicate a specific scale. The maximum y-axis value for all circos plots is 24, as indicated in the figure legend.

   • Line 189: it would also be interesting how many peaks are "conserved" between the different time points studied, so not only to compare the gene lists of closest genes but also the intersecting peaks and then the closest genes to the intersecting peaks. __Response: __We have added this information in Table 7 and in the manuscript starting on Line 203. Using the new dataset of consensus peaks between two replicates, there were 88 genes associated with an SMC3 peak across all three time points, most of which were close to a centromeric region.

   • What is the distribution of the peaks over diverse genetic elements, such as gene bodies, introns, convergent/ divergent/ tandem intergenic regions? In yeast, cohesion is particularly enriched in convergent intergenic regions, so it would be interesting to see how this behaves in P. falciparum. __Response: __We would have liked to define how many peaks were in intergenic versus genic regions of the genome, but the dataset of “genes” from PlasmoDB includes UTRs. Thus, we would need a better annotation of the genome to perform this analysis. Regardless, we calculated the average SMC3 peak enrichment (shared between both replicates) in intergenic regions between convergent and divergent genes (see what is now Supp. Fig. 3B and Table 6). As we now state in the manuscript on line 198, we see a slight enrichment in regions between convergent genes at all time points, but the differences were not significant.

   • Line 130 intra-chromosomal interactions (word missing) __Response: __Thank you for pointing this out. We have corrected this.

   • Contrary to Figure 1D, the WB in Figure 3A indicates strong expression of Smc3 in rings. Please comment on this discrepancy. __Response: __While extracts from all time points were run on the same western blot in Fig. 1D and thus developed for the same amount of time, this was not the case for Fig. 3A. In Fig. 3A, the samples were run on different blots and exposed for different times, so while we can compare SMC3-HA levels between – and + glucosamine for each time point, the levels at 12 hpi cannot be quantitatively compared to those at 24 or 36hpi.

   • What time point after glucosamine addition represents the WB in Fig. 3A? __Response: __The “12hpi” parasites were sampled approximately 108 hours post glucosamine addition and the “24hpi” parasites sampled approximately 120 hours post glucosamine addition. Basically, the parasites were treated with glucosamine for 96 hours, synchronized, and then harvested 12 and 24 hours later.

   • Line 233 / Suppl Figure 3: Isn't it a bit concerning that the untreated control parasites at 24 hpi statistically corresponded to 18-19 hpi? And to what timepoint did the wt parasites correspond? __Response: __We are not concerned by this, and we have included the WT parasites in what is now Supp. Fig. 5B for better comparison. In the analysis presented in Supp. Fig. 5B, regardless of glucosamine presence or absence, the differences among replicates and strains at 12 and 24hpi are, in our opinion, minimal, amounting to one or two hours of the 48-hour IDC. In our extensive experience with RNA-seq across the P. falciparum lDC, this synchronization is extremely tight. As we describe on line 430 of the Materials and Methods, there is a ±3 hour window in our synchronization method, meaning that parasites harvested at 24hpi could be anywhere from 21-27hpi. In addition, the dataset that was used for comparison (from Bozdech et al., 2003) was generated in 2003 in a different laboratory using different strains with microarray. While comparing more recent RNA-seq data to this classic study has become well-established practice and is useful for comparing transcriptional age between replicates and strains, it is inevitable that the calculated “hpi” from (Bozdech et al., 2003) will differ somewhat from our experimental “hpi”. We have indeed seen this small discrepancy in predicted transcriptional age in several of our RNA-seq datasets (unrelated to this study) from trophozoites harvested at 24hpi.

   • Line 264: "whether naturally or via knockdown" - the meaning of this sentence is not entirely clear __Response: __We are referring to depletion of SMC3 at promoters, either naturally (i.e. lack of binding at the promoter at 36hpi that is not the result of SMC3 knockdown, as we show in Fig. 4B) or via SMC3 knockdown, which is not natural but artificial.

   • Figure 4 Legend: A, B, C etc. are mixed up. Response: Thank you for pointing this out. We have corrected this.

   • Figure 4D: the differences seem to be marginally significant, even not significant at all (q=0.8) for gap45 at 12hpi. __Response: __If one defines a significance cutoff of q = 0.05 (as is common practice in differential expression analyses), then the differences are significant. For a small minority of invasion genes (such as gap45), we do observe significance at either 12 hpi or 24 hpi, but not both. Thus, we have removed the word “significant” from the descriptions of each dataset in Tab 1 of what is now Table 18. however, we do not believe that this rules out a role for SMC3 at such a gene during interphase. What is now Table 18 offers a longer list of invasion-related genes, most of which are more “significantly” affected than rap2 and gap45.

   • Figure 4F shows FACS data using SYBR green as a DNA stain. The authors could exploit this data to look at the relative DNA content per cell as a measure of parasite stage, since more mature parasites will have more DNA (mean fluorescence intensity). How did the corresponding parasite cultures look in Giemsa smears? Response: We have repeated our growth curve analysis several times and with more clones and have concluded that there is not a significant growth phenotype in SMC3 KD parasites (see what is now Supp. Fig. 4B). We have added images of Giemsa-stained parasites in untreated and glucosamine-treated parasites at all time points to demonstrate a lack of an obvious morphological phenotype in SMC3 KD parasites (see what is now Supp. Fig. 5A).

   • Are RNAseq replicates biological replicates from independent experiments or technical replicates? __Response: __RNA-seq replicates are technical replicates from the same parasite clone.

   • Why does the number of genes analysed for differential gene expression differ between the comparisons? __Response: __If the reviewer is referring to the discrepancy between the total number of genes for different time points [for example, between what is now Table 9 (12hpi) and Table 10 (24hpi)], this is because in the RNA-seq/differential expression analysis, there have to be reads mapping back to a gene in order for that gene to be included in the analysis. Thus, if a gene is not transcribed at a given time point in the treated or untreated samples, it will not be included in the analysis. Gene transcription fluctuates significantly over the course of the IDC, so different time points will have different total numbers of transcribed genes.

   • Line 372: Do you mean the proteins or the genes? AP2-I has a peak at 24 hpi and 36 hpi, and its interacting AP2 factor Pf3D7_0613800 at all time points. __Response: __We are referring to the genes. With the new ChIP-seq analysis including the second replicate, there are no consensus SMC3 peaks associated with ap2-I, *bdp1, *or Pf3D7_0613800 (see what is now Table 7).

   • Line 480: no aldolase was shown. __Response: __We have removed this sentence.

   • Line 838: include GO analysis in methods __Response: __We have added this.

   Reviewer #2 (Significance (Required)): The paper addresses the function of the cohesin complex in gene regulation of malaria parasites for the first time. Due to the conserved nature of the complex, the data may be interesting for a broad audience of scientists interested in nuclear biology and cell division/ gene regulation.

   *Reviewer #3 *

   (Evidence, reproducibility and clarity (Required)):

   *Summary:

   In the presented manuscript by Rosa et al. the authors investigate the longstanding question of how P. falciparum achieves the tight transcriptional regulation of its genome despite the apparent absence of many canonical sequence specific transcription factor families found in other eukaryotes. To do this the authors investigate the role of the spatial organization of the genome in this context, by performing a functional characterization of the conserved cohesion subunit SMC3 and its putative role in transcriptional regulation in P. falciparum. Using Cas9 mediated genome editing the authors generated a SMC3-3xHA-glmS parasite line, which they subsequently used to show expression of the protein over the asexual replication cycle by western blot and IFA analysis. In addition, using co-IP experiments coupled with mass spectrometry they identified the additional components of the cohesion complex also found in other eukaryotes as interaction partners of SMC3 in the parasite, thereby confirming the presence of the conserved cohesin complex in P. falciparum. By using a combination of ChIP-seq and RNA-seq experiments in SMC3 knockdown parasites the authors furthermore show that a reduction of SMC3 resulted in the up-regulation of a specific set of genes involved in invasion and egress in the early stages of the asexual replication cycle and that this up-regulation in transcription is correlated with a loss of SMC3 enrichment at these genes. From these observations the authors conclude, that SMC3 binds dynamically to a subset of genes and works as a transcriptional repressor, ensuring the timely expression of the bound genes. Overall, the presented data is intriguing, of high quality and very well presented. However, there are some points, which should be addressed to bolster the conclusions drawn by the authors.

   Major points: I was not able to find the deposited datasets in the BioProject database under the given accession number. This should obviously be addressed and would have been nice to be able to have a look at these datasets also for the review process. *__Response: __We apologize for not giving the reviewers access. As the manuscript has been made available as a pre-print (which includes data accession numbers), but has not yet been published, we have not activated access to the data on the database.

   *SMC3-ChIP-seq experiments:

   "168 were bound by SMC3 across all three time points (Fig. 2D). However, most SMC3-bound genes showed a dynamic binding pattern, with a peak present at only one or two time points (Fig. 2B,D)."

   Here it would be interesting to actually have more than one replicate of each of these ChIP-seq time points. This could provide a better idea of how "dynamic" these binding patterns actually are. Furthermore, I was missing a list of these 168 genes, which are constantly bound by SMC3. Anything special about those? What actually happens to this subset of genes in the SMC3 knockdown parasites? Do they show similar transcriptional changes?*

   __Response: __We have now performed a second biological replicate of the SMC3-3HA ChIP-seq with a different clone at all time points. We compared this data to that from the original clone and found significant overlap of the peaks called (see what is now Table 4 and Supp. Fig. 3A). We generated a stringent list of peaks that were shared between both clones at each time point and repeated all downstream analyses (see what are now Tables 5-8). We found that our conclusions were largely unchanged. Text describing these experiments and analyses have been added throughout the results section. Using the new dataset of consensus peaks between two replicates, there were 88 genes associated with an SMC3 peak across all three time points (see what is now Table 7). The genes that are associated with an SMC3 peak at all time points are, in general, those closest to centromeric/pericentromeric regions and show no obvious functional relationship to each other. Out of these 88 genes, four are significantly up- or downregulated at 12 hpi and 26 are significantly up- or downregulated at 24 hpi. The most significantly downregulated of these genes in both datasets is smc3 itself.

   *SMC3-knockdown experiments:

   In Sup. Fig. 1 there is a double band in the HA-western blot in the 2nd cycle -GlcN. sample. This second band is absent in all other HA-western shown. Have the authors any idea where that second band comes from?*

   __Response: __As the reviewer says, we do not see this second band in most of our western blots. It is possible that it is just a small amount of degradation in the lysate.

   In Figure 3A, the WB data shown is slightly contrasting the RNA-seq quantification (3B). The knock-down on protein level seems to be stronger in the 12 hpi samples here than in the 24 hpi samples. Although the band for HA-SMC3 is stronger at the 12 hpi TP there's no band visible in the + GlcN. sample. There's however in the 24 hpi samples. Could the authors comment on this?

   __Response: With regard to the discrepancy of the knockdown and protein versus RNA level, it is quite common for transcript levels to not agree with protein levels. This is why we always confirm a transcriptional knockdown with western blot analysis using appropriate loading controls. We are not sure why there is a more dramatic knockdown of SMC3 at 12hpi than at 24hpi, as these samples came from the same culture, but were simply harvested 12 hours apart. __

   *"Comparison of our RNA-seq data to the time course transcriptomics data from (Painter et al., 2018) revealed that SMC3 depletion at 12 hpi caused downregulation of genes that normally reach their peak expression in the trophozoite stage (18-30 hpi), with the majority of upregulated genes normally reaching their peak expression in the schizont and very early ring stages (40-2 hpi) (Fig. 3E). At 24 hpi, a similar trend is observed, with most downregulated genes normally peaking in expression in trophozoite stage (24-32 hpi) and the majority of upregulated genes peaking in expression at very early ring stage (2 hpi) (Fig. 3F)."

   I'm not fully convinced by these presented results/conclusions. This dataset would greatly benefit from the inclusion of additional later time points.*

   __Response: __To be clear, we are most interested in the transcriptional role of SMC3 during interphase, where results are not confounded by its potential role in mitosis. However, we did collect a 36hpi time point in the SMC3-3HA-glmS and WT strain, with and without glucosamine. We have added this last time point and the WT data from the other two time points to the manuscript (see Tables 11-13). Unfortunately, and for reasons unknown, the WT replicates treated with glucosamine showed a significantly advanced “transcriptional age” compared to the other replicates at 36hpi (see what is now Supp. Fig. 5B). Thus, we did not feel comfortable performing the RNA-seq analysis as we did with the other two time points (i.e. subtracting up- and down-regulated genes from the WT control from the SMC3-3HA-glmS data sets). We have added this information to the results section (Lines 256 and 261). As the WT parasites treated with glucosamine were approximately 8 hours in advance of the untreated WT parasites for the 36hpi time point, any up- and down-regulated genes might have been due to differences in the cell cycle rather than due to glucosamine treatment. The glmS system of inducible knockdown is widely used in P. falciparum; however, to our knowledge, no lab has investigated whether glucosamine treatment affects transcription in wildtype cells over the course of the IDC. Thus, for accurate phenotypic characterization of any protein with this system with regard to transcriptomics, we thought it was important to provide an RNA-seq dataset to define the cohort of genes affected by glucosamine treatment in WT parasites. We hope that our study will demonstrate the importance of using stringent controls when using inducible knockdown systems.

   We performed differential expression analysis of the SMC3-3HA-glmS parasites with and without glucosamine at 36hpi (we have added this data in Table 11). Again, significantly up- and down-regulated genes were not filtered using the WT dataset. With this analysis, we see only three genes from the list of invasion-related genes (Hu et al., 2010) that are up-regulated, but none of them have a significant q-value (Tab 5 of Table 18). Thus, depletion of SMC3 in late stage parasites does not lead to up-regulation of the same genes that are upregulated at 12 and 24hpi. We have added this information to the text (Line 277).

   *The presented upregulation of the egress and invasion related genes is hard to pinpoint to be a direct effect of transcriptional changes due to the SMC3 knockdown. While there's a slight upregulation of these genes they still seem to be regulated in their normal overall transcriptional program as shown in Figure 4D/E. *

   __Response: __We provide evidence of a direct effect of SMC3 binding by combining differential expression analysis performed upon SMC3 knockdown with SMC3 ChIP-seq at corresponding time points. As we show in what is now Fig. 4C and D, promoter accessibility of these egress/invasion genes correlates with their transcriptional activity. However, SMC3 binding to the promoters of these same genes shows inverse correlation with their transcriptional activity (what is now Fig. 4B and D). While we believe that SMC3 does contribute to the repression of these genes at specific time points during the cell cycle, it is highly likely that SMC3 is just one protein of many that regulates these genes. Moreover, and especially since we do not see a growth phenotype in the SMC3 KD, it is possible that another protein or even SMC1 could compensate for loss of SMC3 at these promoter regions. We now state these possibilities on lines 346 383 of the Discussion.

   *So the changes could in theory also be explained by the differences in cell cycle progression which are present between +/- GlcN. cultures (Sup. Fig. 3). The presented normalization to the microarray data is a well-established practice to correct for this but, as presented seems to have its limitation with these parasite lines (line 233, glucosamine treated parasites harvested at 24 hpi correspond statistically to approximately 18-19 hpi (Supp. Fig. 3).) *

   __Response: __In the analysis presented in what is now Supp. Fig. 5B, regardless of glucosamine presence or absence, the differences among replicates and strains at 12 and 24hpi are, in our opinion, minimal, amounting to one or two hours of the 48-hour IDC. In our extensive experience with RNA-seq across the P. falciparum lDC, this synchronization is extremely tight. As we describe on lines 416-421 of the Materials and Methods, there is a ±3 hour window in our synchronization method, meaning that parasites harvested at 24hpi could be anywhere from 21-27hpi. In addition, the dataset that was used for comparison (from Bozdech et al., 2003) was generated in 2003 in a different laboratory using different strains with microarray. While comparing more recent RNA-seq data to this classic study has become well-established practice and is useful for comparing transcriptional age between replicates and strains, it is inevitable that the calculated “hpi” from (Bozdech et al., 2003) will differ somewhat from our experimental “hpi”. We have indeed seen this small discrepancy in predicted transcriptional age in several of our RNA-seq datasets from trophozoites harvested at 24hpi.

   By including additional later time points, one could actually follow the expression profiles over the whole cycle and elucidate if there's an actual transcriptional up-regulation of the genes, or if the + GlcN. parasites show a faster cell cycle progression, with a shifted peak expression timing compared to the - GlcN. parasites. __Response: __We did collect a 36hpi time point in the SMC3-3HA-glmS and WT strain, with and without glucosamine. We have added this last time point and the WT data from the other two time points to what is now Supp. Fig. 5. Unfortunately, and for reasons unknown, the WT replicates treated with glucosamine showed a significantly advanced “transcriptional age” compared to the other replicates at 36hpi. Thus, we did not feel comfortable performing the RNA-seq analysis as we did with the other two time points (i.e. subtracting up- and down-regulated genes from the WT control from the SMC3-3HA-glmS data sets). We have added this information to the results section (Lines 256 and 261). As the WT parasites treated with glucosamine were approximately 8 hours in advance of the untreated WT parasites for the 36hpi time point, any up- and down-regulated genes might have been due to differences in the cell cycle rather than due to glucosamine treatment. The glmS system of inducible knockdown is widely used in P. falciparum; however, to our knowledge, no lab has investigated whether glucosamine treatment affects transcription in wildtype cells over the course of the IDC. Thus, for accurate phenotypic characterization of any protein with this system with regard to transcriptomics, we thought it was important to provide an RNA-seq dataset to define the cohort of genes affected by glucosamine treatment in WT parasites. We hope that our study will demonstrate the importance of using stringent controls when using inducible knockdown systems.

   *"These genes show SMC3 enrichment at their promoter regions at 12 and 24 hpi, but not at 36 hpi (Fig. 4C), and depletion of SMC3 resulted in upregulation at both 12 and 24 hpi (Fig. 4D). Comparison of the SMC3 ChIP-seq data with published Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) data (Toenhake et al., 2018) and mRNA dynamics data (Painter et al., 2018) from similar time points in the IDC revealed that SMC3 binding at the promoter regions of these genes inversely correlates with chromatin accessibility (Fig. 4C) and their mRNA levels (Fig. 4E), which both peak in schizont stages. These data are consistent with a role of SMC3 in repressing this gene subset until their appropriate time of expression in the IDC."

   The presented correlations certainly make an intriguing point towards the authors conclusion that SMC3/cohesin depletion from the promoter regions of the genes results in a de-repression of these genes and their transcriptional activation. However, the SMC3 knockdown is not complete and only up to 69% as presented on RNA level in these parasites. Therefore a control experiment which needs to be done is to actually show the loss of SMC3 from the presented activated example genes in the knockdown parasites. This could easily be done by ChIP-qPCR or even ChIP-seq, to get a global picture of the actual changes in SMC3 occupation in the knockdown parasites in correlation with changes in transcript levels. *__Response: __While SMC3-3HA-glmS knockdown is not complete at the RNA level, it is fairly robust at the protein level, especially at 12hpi (Fig. 3A).

   *"These data suggest that SMC3 knockdown results in a faster progression through the cell cycle or a higher rate of egress/invasion."

   The authors could greatly strengthen their conclusions by investigating this thoroughly. Pinpointing the observed phenotype to an actual increase in invasion or egress would add to the authors main conclusion that the loss of SMC3 de-regulates the timing of gene expression for these invasion related genes thereby increasing their transcript levels and thus leading to a higher rate of egress/invasion. To determine cell cycle progression simple comparisons between DNA content using a flow cytometer at timepoints together with visual inspection of Giemsa stained blood smears would give a ggod indication towards changes in cell cycle progression. In addition invasion/egress assays by counting newly invaded rings per schizont could reveal, if there are changes in the rate of egress/invasion upon SMC3 knockdown.*

   __Response: We have repeated our growth curve analysis several times and with more clones and have concluded that there is not a significant growth phenotype in SMC3 KD parasites (see what is now Supp. Fig. 4B). We have added images of Giemsa-stained parasites from the knockdown time course we performed to what is now Supp. Fig. 5A. We see no obvious differences in cell morphology caused by glucosamine treatment in the WT or SMC3-3HA-glmS parasites. As we discuss on line 327, very little intact cohesin complex seems to be required for normal growth and mitosis in S. cerevisiae and D. melanogaster, which is probably why we do not see an obvious growth or morphological phenotype. We believe that SMC3 is probably only a part of a complex controlling transcription of these invasion or egress genes. Thus, the up-regulation of these genes upon SMC3 KD might not be enough to lead to a significant growth or invasion phenotype. __

   *Minor points:

   In the MM section on the Cas9 experiments it says dCas9 where it should be Cas9 (line 425)*

   __Response: __Thank you for pointing this out. We have corrected this.

   It would be great to add which HP1 antibody was used in which dilution in the IFAs to the MM section. __Response: __We have added this information to the Materials and Methods section.

   In Figure 4C for the gap45 gene there's is some green peak floating around which should not be there. __Response: __Thank you for pointing this out, we have corrected it.

   *Reviewer #3 (Significance (Required)):

   Significance: The manuscript investigates a very timely topic by trying to uncover new molecular mechanisms of transcriptional regulation in P. falciparum. Investigating the role of the cohesin complex/SMC3 in this context provides valuable new insights to the field. While the first part with the description of the SMC3 cell line and the co-IP experiments largely confirms published data on the existence and composition of the cohesin complex in Plasmodium and its enrichment at the centromeres, the second part is especially intriguing since it investigates the molecular function of SMC3 in more detail. The results pointing to a role of SMC3/cohesin as a transcriptional repressor are of great interest to the field and will open up new concepts for future investigation.*

   *Audience: The work is particularly interesting for people interested in gene regulatory processes in Plasmodium and Apicomplexan parasites in general. At the same time it also nicely points towards shared principles of gene regulation to other eukaryotes in relation to the spatial organization of the genome making the work also very interesting for a broader audience with interest in the general principles of gene regulatory processes in eukaryotic organisms.

   Expertise: P. falciparum epignetics and chromatin biology / gene regulation / Cas9 gene editing*

   CROSS-CONSULTATION COMMENTS

   All reviewers agree that the paper addresses an important topic and provides convincing evidence for enrichment of the cohesin component Smc3 at P. falciparum centromers. In contrast, evidence for a function of Smc3 as a transcriptional repressor of genes in the first part of the parasite life cycle is less well supported. All reviewers agree that the statistical significance of the ChIP experiments needs to be impoved by including biological replicates. In addition, the phenotype of the conditional knock-down should be analysed in more detail by clarifying whether faster cell cycle progression or higher invasion rate are responsible for the observed growth adavantage. Inclusion of transcriptional data from a later time point in addition to the presented data for 12 hpi and 24 hpi was also requested by all reviewers. Finally, several inconsistencies require clarification.

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   Referee #3

   Evidence, reproducibility and clarity

   Summary:

   In the presented manuscript by Rosa et al. the authors investigate the longstanding question of how P. falciparum achieves the tight transcriptional regulation of its genome despite the apparent absence of many canonical sequence specific transcription factor families found in other eukaryotes. To do this the authors investigate the role of the spatial organization of the genome in this context, by performing a functional characterization of the conserved cohesion subunit SMC3 and its putative role in transcriptional regulation in P. falciparum.

   Using Cas9 mediated genome editing the authors generated a SMC3-3xHA-glmS …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   Referee #3

   Evidence, reproducibility and clarity

   Summary:

   In the presented manuscript by Rosa et al. the authors investigate the longstanding question of how P. falciparum achieves the tight transcriptional regulation of its genome despite the apparent absence of many canonical sequence specific transcription factor families found in other eukaryotes. To do this the authors investigate the role of the spatial organization of the genome in this context, by performing a functional characterization of the conserved cohesion subunit SMC3 and its putative role in transcriptional regulation in P. falciparum.

   Using Cas9 mediated genome editing the authors generated a SMC3-3xHA-glmS parasite line, which they subsequently used to show expression of the protein over the asexual replication cycle by western blot and IFA analysis. In addition, using co-IP experiments coupled with mass spectrometry they identified the additional components of the cohesion complex also found in other eukaryotes as interaction partners of SMC3 in the parasite, thereby confirming the presence of the conserved cohesin complex in P. falciparum. By using a combination of ChIP-seq and RNA-seq experiments in SMC3 knockdown parasites the authors furthermore show that a reduction of SMC3 resulted in the up-regulation of a specific set of genes involved in invasion and egress in the early stages of the asexual replication cycle and that this up-regulation in transcription is correlated with a loss of SMC3 enrichment at these genes. From these observations the authors conclude, that SMC3 binds dynamically to a subset of genes and works as a transcriptional repressor, ensuring the timely expression of the bound genes.

   Overall, the presented data is intriguing, of high quality and very well presented. However, there are some points, which should be addressed to bolster the conclusions drawn by the authors.

   Major points:

   I was not able to find the deposited datasets in the BioProject database under the given accession number. This should obviously be addressed and would have been nice to be able to have a look at these datasets also for the review process.

   SMC3-ChIP-seq experiments:

   "168 were bound by SMC3 across all three time points (Fig. 2D). However, most SMC3-bound genes showed a dynamic binding pattern, with a peak present at only one or two time points (Fig. 2B,D)."

   Here it would be interesting to actually have more than one replicate of each of these ChIP-seq time points. This could provide a better idea of how "dynamic" these binding patterns actually are. Furthermore, I was missing a list of these 168 genes, which are constantly bound by SMC3. Anything special about those? What actually happens to this subset of genes in the SMC3 knockdown parasites? Do they show similar transcriptional changes?

   SMC3-knockdown experiments:

   In Sup. Fig. 1 there is a double band in the HA-western blot in the 2nd cycle -GlcN. sample. This second band is absent in all other HA-western shown. Have the authors any idea where that second band comes from?

   In Figure 3A, the WB data shown is slightly contrasting the RNA-seq quantification (3B). The knock-down on protein level seems to be stronger in the 12 hpi samples here than in the 24 hpi samples. Although the band for HA-SMC3 is stronger at the 12 hpi TP there's no band visible in the + GlcN. sample. There's however in the 24 hpi samples. Could the authors comment on this?

   "Comparison of our RNA-seq data to the time course transcriptomics data from (Painter et al., 2018) revealed that SMC3 depletion at 12 hpi caused downregulation of genes that normally reach their peak expression in the trophozoite stage (18-30 hpi), with the majority of upregulated genes normally reaching their peak expression in the schizont and very early ring stages (40-2 hpi) (Fig. 3E). At 24 hpi, a similar trend is observed, with most downregulated genes normally peaking in expression in trophozoite stage (24-32 hpi) and the majority of upregulated genes peaking in expression at very early ring stage (2 hpi) (Fig. 3F)."

   I'm not fully convinced by these presented results/conclusions. This dataset would greatly benefit from the inclusion of additional later time points. The presented upregulation of the egress and invasion related genes is hard to pinpoint to be a direct effect of transcriptional changes due to the SMC3 knockdown. While there's a slight upregulation of these genes they still seem to be regulated in their normal overall transcriptional program as shown in Figure 4D/E. So the changes could in theory also be explained by the differences in cell cycle progression which are present between +/- GlcN. cultures (Sup. Fig. 3). The presented normalization to the microarray data is a well-established practice to correct for this but, as presented seems to have its limitation with these parasite lines (line 233, glucosamine treated parasites harvested at 24 hpi correspond statistically to approximately 18-19 hpi (Supp. Fig. 3).) By including additional later time points, one could actually follow the expression profiles over the whole cycle and elucidate if there's an actual transcriptional up-regulation of the genes, or if the + GlcN. parasites show a faster cell cycle progression, with a shifted peak expression timing compared to the - GlcN. parasites.

   "These genes show SMC3 enrichment at their promoter regions at 12 and 24 hpi, but not at 36 hpi (Fig. 4C), and depletion of SMC3 resulted in upregulation at both 12 and 24 hpi (Fig. 4D). Comparison of the SMC3 ChIP-seq data with published Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) data (Toenhake et al., 2018) and mRNA dynamics data (Painter et al., 2018) from similar time points in the IDC revealed that SMC3 binding at the promoter regions of these genes inversely correlates with chromatin accessibility (Fig. 4C) and their mRNA levels (Fig. 4E), which both peak in schizont stages. These data are consistent with a role of SMC3 in repressing this gene subset until their appropriate time of expression in the IDC."

   The presented correlations certainly make an intriguing point towards the authors conclusion that SMC3/cohesin depletion from the promoter regions of the genes results in a de-repression of these genes and their transcriptional activation. However, the SMC3 knockdown is not complete and only up to 69% as presented on RNA level in these parasites. Therefore a control experiment which needs to be done is to actually show the loss of SMC3 from the presented activated example genes in the knockdown parasites. This could easily be done by ChIP-qPCR or even ChIP-seq, to get a global picture of the actual changes in SMC3 occupation in the knockdown parasites in correlation with changes in transcript levels.

   "These data suggest that SMC3 knockdown results in a faster progression through the cell cycle or a higher rate of egress/invasion."

   The authors could greatly strengthen their conclusions by investigating this thoroughly. Pinpointing the observed phenotype to an actual increase in invasion or egress would add to the authors main conclusion that the loss of SMC3 de-regulates the timing of gene expression for these invasion related genes thereby increasing their transcript levels and thus leading to a higher rate of egress/invasion. To determine cell cycle progression simple comparisons between DNA content using a flow cytometer at timepoints together with visual inspection of Giemsa stained blood smears would give a ggod indication towards changes in cell cycle progression. In addition invasion/egress assays by counting newly invaded rings per schizont could reveal, if there are changes in the rate of egress/invasion upon SMC3 knockdown.

   Minor points:

   In the MM section on the Cas9 experiments it says dCas9 where it should be Cas9 (line 425)

   It would be great to add which HP1 antibody was used in which dilution in the IFAs to the MM section.

   In Figure 4C for the gap45 gene there's is some green peak floating around which should not be there.

   Significance

   The manuscript investigates a very timely topic by trying to uncover new molecular mechanisms of transcriptional regulation in P. falciparum. Investigating the role of the cohesin complex/SMC3 in this context provides valuable new insights to the field. While the first part with the description of the SMC3 cell line and the co-IP experiments largely confirms published data on the existence and composition of the cohesin complex in Plasmodium and its enrichment at the centromeres, the second part is especially intriguing since it investigates the molecular function of SMC3 in more detail. The results pointing to a role of SMC3/cohesin as a transcriptional repressor are of great interest to the field and will open up new concepts for future investigation.

   Audience:

   The work is particularly interesting for people interested in gene regulatory processes in Plasmodium and Apicomplexan parasites in general. At the same time it also nicely points towards shared principles of gene regulation to other eukaryotes in relation to the spatial organization of the genome making the work also very interesting for a broader audience with interest in the general principles of gene regulatory processes in eukaryotic organisms.

   Expertise:

   P. falciparum epignetics and chromatin biology / gene regulation / Cas9 gene editing

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   Referee #2

   Evidence, reproducibility and clarity

   Rosa et al, Review Commons

   The manuscript by Rosa et al. addresses the function of the cohesion subunit Smc3 in gene regulation during the asexual life cycle of P. falciparum. Cohesin is a conserved protein complex involved in sister chromatin cohesion during mitosis and meiosis in eukaryotic cells. Cohesin also modulates transcription and DNA repair by mediating long range DNA interactions and regulating higher order chromatin structure in mammals and yeast. In P. falciparum, the Cohesin complex remains largely uncharacterized. In this manuscript, the authors present mass spectrometry data from co-IPs showing that Smc3 interacts …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   ---

   Referee #2

   Evidence, reproducibility and clarity

   Rosa et al, Review Commons

   The manuscript by Rosa et al. addresses the function of the cohesion subunit Smc3 in gene regulation during the asexual life cycle of P. falciparum. Cohesin is a conserved protein complex involved in sister chromatin cohesion during mitosis and meiosis in eukaryotic cells. Cohesin also modulates transcription and DNA repair by mediating long range DNA interactions and regulating higher order chromatin structure in mammals and yeast. In P. falciparum, the Cohesin complex remains largely uncharacterized. In this manuscript, the authors present mass spectrometry data from co-IPs showing that Smc3 interacts with Smc1 and a putative Rad21 orthologue (Pf3D7_1440100, consistent with published data from Batugedara et al and Hilliers et al), as well as a putative STAG domain protein orthologue (PF3D7_1456500). Smc3 protein appears to be most abundant in schizonts, but ChIPseq indicates predominant enrichment of Smc3 in centromers in ring and trophozoite stages. In addition, Smc3 dynamically binds with low abundance to other loci across the genome; however, the enrichment is rather marginal and only a single replicate was conducted for each time point making the data interpretation difficult. Conditional knock-down using a GlmS ribozyme approach indicates that parasites with reduced levels of Smc3 have a mild growth advantage, which is only evident after five asexual replication cycles and which the authors attribute to the transcriptional upregulation of invasion-linked genes following Smc3 KD. Indeed, Smc3 seems to be more enriched upstream of genes that are upregulated after Smc3 KD in rings than in downregulated genes, indicating that Smc3/cohesin may have a function in supressing transcription of these schizont specific genes until they are needed. The manuscript is concise and very well written, however it suffers from the lack of experimental replicates for ChIP experiments and a better characterization of the phenotype of conditional KD parasites.

   Major comments

   • In the mass spectrometry analysis, many seemingly irrelevant proteins are identified at similar abundance to the putative rad21 and ssc3 orthologues, and therefore the association with the cohesion complex seems to be based mostly on analogy to other species rather than statistical significance. Hence, it would be really nice to see a validation of the novel STAG domain and Rad21 proteins, for example by Co-IP using double transgenic parasites.
   • The ChIPseq analysis presented here is based on single replicates for each of the three time points. The significance cutoffs for the peaks are rather high (q < 0.05). Therefore, the relevance of the marginally enriched dynamic peaks (average relative enrichment of <1.2 fold for genes upregulated in rings 12 hpi in Figure 4A and B) does not appear to be very robust. Even in ChIPseq experiments using non-immune IgG, hundreds of peaks are usually called with MACS2 with a similar magnitude. So, to substantiate the data for extra-centromeric peaks convincingly, replicates and more stringent statistics are necessary. In addition, the authors should also compare their data to published PfSmc3 ChIP data from Batugedara et al 2020 (GSE116219).
   • The authors argue that during schizogony, cohesin may no longer be required at centromers, explaining the low ChIPsignal at this stage (Line 301). However, during schizogony parasites undergo repeated rounds of DNA replication (S-phase) and mitosis (M-phase) to generate multinucleated parasites; and concentrated spots of Smc3 are observed in each nucleus in schizonts by IFA. In turn, the strong presence of Smc3 at centromers in ring stage parasites is surprising, particularly since the Western Blot in Figure 1D shows most expression of Smc3 in schizonts and least in rings; and Smc3 is undetectable in rings by IFA. Yet, the ChIP signal shows very strong enrichment at centromers, long before S phase produces sister chromatids. What could be the reason for this discrepancy? Again, ChIP replicates and controls would be helpful in distinguishing technical problems with the ChIP from biologically relevant differences.
   • It is surprising that a conserved protein like Smc3 shows such a subtle phenotype, given that it is predicted to be essential and its orthologues have a function in mitosis. Generally, only limited data are presented to characterize the Smc3 KD parasites, and more detail should be included. For example validation of the parasite line using a PCR screen for integration and absence of wt, parasite morphology after KD, and/or analysis of the KD parasites for cell cycle status.
   • Synchronization was performed at the beginning of the growth time course, which would be expected to result in a stepwise increase in parasitemia every 48 hours; however, the parasitemia according to Fig. 4F rises steadily, which would indicate that the parasites are actually not very synchronous.
   • The question of whether Smc3 causes a shorter parasite life cycle (quicker progression) or more invasion is important and could be experimentally addressed by purifying synchronous schizont stage parasites and determining their invasion rates as well as morphological examination of the Giemsa smears over the time course.
   • Please also compare Smc3 transcriptional levels in transgenic parasites to those in wt parasites to rule out that the genetic modification has lead to artificial upregulation of Smc3 transcription.
   • According to Figure S2, even more genes were deregulated at the 12 hpi time point in the WT parasites than in Smc3 parasites, and even to a much higher extent. What "transcriptional age" did the WT control parasites have at each time point?
   • A negative correlation with transcription is well established in S. cerevisiae, particularly at inducible genes. How does Smc3 enrichment generally look like for genes that show maximal expression at each of the time point?
   • Line 590: according to the methods, a 36 hpi KD time point was also harvested. Why are the data not shown/analysed?

   Minor Comments

   • Line 103/104: the hinge domain and ATPase head domain are mentioned, please annotate these in Figure 1A.
   • Figure 1D: the kDa scale is missing from the H3 WB.
   • What is the scale indicated by different colors in Fig. 2A?
   • Line 189: it would also be interesting how many peaks are "conserved" between the different time points studied, so not only to compare the gene lists of closest genes but also the intersecting peaks and then the closest genes to the intersecting peaks.
   • What is the distribution of the peaks over diverse genetic elements, such as gene bodies, introns, convergent/ divergent/ tandem intergenic regions? In yeast, cohesion is particularly enriched in convergent intergenic regions, so it would be interesting to see how this behaves in P. falciparum.
   • Line 130 intra-chromosomal interactions (word missing)
   • Contrary to Figure 1D, the WB in Figure 3A indicates strong expression of Smc3 in rings. Please comment on this discrepancy.
   • What time point after glucosamine addition represents the WB in Fig. 3A?
   • Line 233 / Suppl Figure 3: Isn't it a bit concerning that the untreated control parasites at 24 hpi statistically corresponded to 18-19 hpi? And to what timepoint did the wt parasites correspond?
   • Line 264: "whether naturally or via knockdown" - the meaning of this sentence is not entirely clear
   • Figure 4 Legend: A, B, C etc. are mixed up.
   • Figure 4D: the differences seem to be marginally significant, even not significant at all (q=0.8) for gap45 at 12hpi.
   • Figure 4F shows FACS data using SYBR green as a DNA stain. The authors could exploit this data to look at the relative DNA content per cell as a measure of parasite stage, since more mature parasites will have more DNA (mean fluorescence intensity). How did the corresponding parasite cultures look in Giemsa smears?
   • Are RNAseq replicates biological replicates from independent experiments or technical replicates?
   • Why does the number of genes analysed for differential gene expression differ between the comparisons?
   • Line 372: Do you mean the proteins or the genes? AP2-I has a peak at 24 hpi and 36 hpi, and its interacting AP2 factor Pf3D7_0613800 at all time points.
   • Line 480: no aldolase was shown.
   • Line 838: include GO analysis in methods

   Referees cross-commenting

   All reviewers agree that the paper addresses an important topic and provides convincing evidence for enrichment of the cohesin component Smc3 at P. falciparum centromers. In contrast, evidence for a function of Smc3 as a transcriptional repressor of genes in the first part of the parasite life cycle is less well supported. All reviewers agree that the statistical significance of the ChIP experiments needs to be impoved by including biological replicates. In addition, the phenotype of the conditional knock-down should be analysed in more detail by clarifying whether faster cell cycle progression or higher invasion rate are responsible for the observed growth adavantage. Inclusion of transcriptional data from a later time point in addition to the presented data for 12 hpi and 24 hpi was also requested by all reviewers. Finally, several inconsistencies require clarification.

   Significance

   The paper addresses the function of the cohesin complex in gene regulation of malaria parasites for the first time. Due to the conserved nature of the complex, the data may be interesting for a broad audience of scientists interested in nuclear biology and cell division/ gene regulation.

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4. 

   Review Commons

   Feb 28, 2023

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   Referee #1

   Evidence, reproducibility and clarity

   Summary

   The present work by Rosa et al., provides convincing data about the presence and functional relevance of the cohesin complex in Plasmodium falciparum blood stages. In accordance with other organisms, the composition of the cohesin complex containing SMC1, SMC3 RAD21 and putatively STAG could be confirmed via pulldown and mass spectrometry. Basic characterization of endogenous tagged SMC3 demonstrated the expression and nuclear localization during IDC, as well as the relatively stable accumulation at centromeric regions, consistent with the known cohesin function in chromatid separation. Furthermore, dynamic and …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   Summary

   The present work by Rosa et al., provides convincing data about the presence and functional relevance of the cohesin complex in Plasmodium falciparum blood stages. In accordance with other organisms, the composition of the cohesin complex containing SMC1, SMC3 RAD21 and putatively STAG could be confirmed via pulldown and mass spectrometry. Basic characterization of endogenous tagged SMC3 demonstrated the expression and nuclear localization during IDC, as well as the relatively stable accumulation at centromeric regions, consistent with the known cohesin function in chromatid separation. Furthermore, dynamic and stage-dependent binding to intergenic regions observed in ChIPseq and major transcriptome aberrations upon knockdown of SMC3 (<70% mRNA reduction via glmS-system) suggest an important function in transcriptional regulation. The underlying mechanism of gene regulation by cohesin via the creation of chromatin clusters or DNA loops as well as its specificity to target sites remain speculation.

   Major criticism

   • Accuracy of ChIPseq with only one replicate per time point (and lacking negative controls without antibody) is not convincing. The authors should include more replicates.
   • Proposed mechanism of repressive effect of SMC3 early in IDC on genes, that get de-repressed in late stages: To claim this mode of function, it would be necessary to include a KD on late stage parasites. If there is an early repressive role of SMC3, upregulated genes should not be affected by late SMC3-KD. Furthermore, the hypothesized repressive effect of SMC3 does not explain the numerous genes downregulated in KD.
   • Due to the fact, that the KD was induced at the exact same timepoint and analysed 12h and 24h after induction it is possible that identified, differentially expressed genes at 24h are not directly regulated by SMC3, but rather due to a general deregulation of gene expression. Did the authors attempt to analyse gene expression upon induction at ring, trophozoite and schizont stage?
   • Based on rapid parasite growth, the authors hypothesize a higher invasion rate due to upregulation of invasion genes. This hypothesis is not supported by quantitative invasion assays or quantification of invasion factors on the protein level. An alternative explanation could be a shorter cell cycle (<48h), as the different cell cycle progression estimation of KD/WT could indicate (SuppFigure 3). Giemsa-stain images of KD/WT parasites should be included to show normal stage development over time.
   • Correlation of SMC3-occupancy/ATAC/expression profile of the exemplary genes rap2 and gap45 (Figure 4C,D,E): is this representative for all upregulated genes?
   • Given that SMC3 appears to be not essential for parasite growth, the authors could generate a null mutant for SMC3, which might allow for easier analysis of differences in gene regulation, cell cycle progression and/or invasion efficiency.

   Significance

   Own opinion

   The authors provide a basic characterization of the cohesin component SMC3 using NGS methods to investigate chromatin binding sites and its potential influence on gene expression. The localisation of SMC3 at centromers as described previously (Batugedara 2020) was confirmed. However, the dynamic binding to other regions in the genome, potentially mediated by other proteins, could not be resolved unequivocal with only one replicate of ChIPseq per time point. Similarly, the RNAseq data demonstrate the relevance of SMC3 for gene expression, but no clear picture of a regulatory mechanism can be drawn at his point. Lacking information about the mode of binding as well as the setup of transcriptome analysis (only two time-shifted sampling points after simultaneous glmS treatment for 96h resulting in incomplete knockdown) cannot definitely elucidate, if SMC3/cohesin is a chromatin factor that affects transcription of genes in general or a specific repressor of stage-specific genes.

   The work will be interesting to a general audience, interested in gene regulation and chromatin remodelling

   The reviewers are experts in Plasmodium cell biology and epigenetic regulation.

   Read the original source
5. 

   Version published to 10.1101/2022.07.21.500927v1 on bioRxiv

   Jul 21, 2022