Architecture, Chromatin and Gene Organization of Toxoplasma gondii Subtelomeres

Round 1

Reviewer 1 Report

Overall a very carefully performed cartography of the genetic landscape of the Toxoplasma gondii subtelomeric regions (ST).  The authors show interstrain plasticiti of the ST regions. Subtelomeric regions are much more important in DNA/Histone research as previously thought. The paper presents a source of information for the small scientific community working this field.

The introduction is very well written, brings you up to the current sand of knowledge. the results are well presented (time consuming to go through), and the discussion is concise.

I have no major or minor criticisms to  advance.

Author Response

Dear, thank you very much for your critical reading and positive evaluation of the manuscript. Sergio Angel

Reviewer 2 Report

Revision of the manuscript by Contreras et al., entitled “Architecture, chromatin and gene organization of Toxoplasma gondii subtelomeres” submitted to Epigenomes.

This manuscript reports the analysis of subtelomeric (ST) regions of the apicomplexan pathogen T. gondii. The main data corresponds to the strain Me49. While usually depleted of genes and other functional elements, ST regions can have biological relevance in some organisms. These regions are often overlooked and excluded from whole-genome studies. The conceptual basis of this study is important because the properties and roles of ST regions in apicomplexan parasites are largely unknow.

The most informative results of the study come from the epigenomic and the transcriptomic analyses. I think the manuscript should be reframed to emphasize these two aspects while removing or summarizing some of the content about the fam genes.  

Specific comments:

The figures need more resolution. Figs 1 and 4 are almost impossible to read in the print version. Zooming in on-screen doesn’t help as it quickly pixelates.

Lines 167-169: Confusing, please rephrase for clarity. What is more pronounced?

Sections 2.3-2.7 are too long for the amount and quality if the information. 2.3: The description and analysis of sat dna in ST is superficial and confusing. The definition of synteny employed is not clear and seems to be confused with presence/absence between chromosomes. Synteny is supposed to define conservation in (either presence or order, depending on the usage of the word) of loci between species/populations, not between chromosomes in an organism. Patterns of Sat DNA in ST should be discussed in reference to the presence, abundance and density of Sat DNAs in the euchromatin. We don’t know if these Sat DNA elements are exclusive to ST or if their abundance and distribution is different from the euchromatin. What’s the spatial relationship between SatDNA blocks and genes in the ST and euch.? Any evidence from expression data that SatDNAs may drive or somehow regulate expression? 2.4: “single genes” is unclear, please clarify what it means in this context. It is important to note that the number of genes in ST regions is not very large, therefore expressing data only in percentages or proportions can be misleading. For example, in figure 3, the slices of the pie charts and the columns should show absolute numbers. The identities of the FA genes in ST could be shown in the manuscript rather than in supplementary data. I would rather see them instead of some of the data shown in tables 2 and 3. Perhaps tables 2 and 3 can be unified and summarized to show only essential information and add the names of FA genes. I’d like to know if the FA genes are single copy or if there are copies of them in the euchromatin. Are you sure the FA copies in ST are not pseudogenes? 2.5: The analysis of FamB and FamC is also superficial. I suggest 3 alternatives: make it more detailed and comprehensive so that we learn more about these proteins than what we already know, make it more concise, or eliminate it from the manuscript. For option 1, you need to go beyond what was done in Lorenzi et al. (2016) regarding protein features, potential functional domains and potential homologues in other species. There are more sophisticated and sensitive methods that were not used here, such as HMM profiles-based searches, searching for deep homologues using structural predictions, etc. 2.6: As conceived, this sequence analysis is not informative. To be aligned together, Fams A, B and C should be known to be homologous (i.e., have some level of sequence similarity. Aligning unrelated sequences doesn’t make sense, and any phylogenetic reconstruction would be uninformative. Even if they were related, a tree with members of the 3 fams and no outgroups is by definition unrooted, so that no inferences about “what came first” can be made. It may be worth doing an alignment between FamB and C if they share some similarity. If the authors want to include these analysis, I’d like to see a list of accession numbers for the sequences employed, an alignment figure as supplementary data and details of how the tree/s was/were inferred. For protein sequence alignment I prefer using a more modern program, such as MUSCLE or MAFFT instead of Clustal. 2.7: Gene family size results from a combination of gene gains and loses, and it is very hard to determine the exact course of events along a phylogeny, especially when the number of genes and species is low, as in this case. I think it’s safe to suggest (although not to conclude) that FamB and FamC expanded in the lineage leading to T. gondii, but one shouldn’t say the same for H. hammondi. I’m not sure that figure 5 is worth showing in the manuscript. Same for figure 7B.

Line 626: the word ‘idea’ is incorrectly used (I see that it translates like that from the mode it’s used in another language). It should be “a more detailed picture of…”

 

 

 

Author Response

Dear, Thank you very much for your critical reading and positive evaluation of the manuscript. We also would like to acknowledge your comments, all of which helped to improve the manuscript significantly.  

Below you will find a detailed response to the reviewers' comments. Changes are highlighted in red font.

 

Sincerely yours

 

Dr. Sergio Angel

 

Specific comments:

The figures need more resolution. Figs 1 and 4 are almost impossible to read in the print version. Zooming in on-screen doesn’t help as it quickly pixelates.

 

Response: Both figures were redone, improving the resolution. Figure 4 is now Figure 5.

Lines 167-169: Confusing, please rephrase for clarity. What is more pronounced?

Response; It was replaced by evident (line 134)

Sections 2.3-2.7 are too long for the amount and quality if the information. 2.3: The description and analysis of sat dna in ST is superficial and confusing.

Response: In this revised version we have done some additional analysis based on the reviewer's suggestions. See below

 

The definition of synteny employed is not clear and seems to be confused with presence/absence between chromosomes. Synteny is supposed to define conservation in (either presence or order, depending on the usage of the word) of loci between species/populations, not between chromosomes in an organism.

Response: We separate this analysis in a new section 2.4. Likewise, we rewrite the synteny analysis.

 

 

 

Patterns of Sat DNA in ST should be discussed in reference to the presence, abundance and density of Sat DNAs in the euchromatin. We don’t know if these Sat DNA elements are exclusive to ST or if their abundance and distribution is different from the euchromatin.

Response: We appreciate the comment. The analysis was incorporated into section 2.3 and figure S3.

What’s the spatial relationship between SatDNA blocks and genes in the ST and euch.?

Response: we appreciate the comment. We did the analysis as shown in section 2.4, figures 3 and FigS4.

 

Any evidence from expression data that SatDNAs may drive or somehow regulate expression?

Response: We have not performed any analysis on this matter. We consider that the detection of ST RNAs not related to genes deserves an in-depth study to confirm their existence. We do refer to the topic of another publication (ref. 30), where they detect miRNAs in sat350 and sat529. We mention this at the end of the conclusions.

 

2.4: “single genes” is unclear, please clarify what it means in this context.

Response: This section became 2.5. We appreciate this clarification. We analyzed the existence of paralogs for each of the genes. The data was incorporated into Table S2 and in the text of section 2.5. Based on that, we ruled out the “single genes” concept.

 

It is important to note that the number of genes in ST regions is not very large, therefore expressing data only in percentages or proportions can be misleading. For example, in figure 3, the slices of the pie charts and the columns should show absolute numbers.

Response: Thank you very much for the comment, the graphs were arranged according to what was suggested (Fig 4).

The identities of the FA genes in ST could be shown in the manuscript rather than in supplementary data. I would rather see them instead of some of the data shown in tables 2 and 3. Perhaps tables 2 and 3 can be unified and summarized to show only essential information and add the names of FA genes. I’d like to know if the FA genes are single copy or if there are copies of them in the euchromatin.

 

Response: The table has been redone based on your comments, and is now Table 2. We only found FA paralogs for the OTU gene. That was fixed in table S2 and mentioned in the text, section 2.5.

Are you sure the FA copies in ST are not pseudogenes?

Response: There is only one FA gene that does not show evidence of expression by transcriptomics and mass spectrometry. A clarification was made in section 2.5, as a separate paragraph.

 

2.5: The analysis of FamB and FamC is also superficial. I suggest 3 alternatives: make it more detailed and comprehensive so that we learn more about these proteins than what we already know, make it more concise, or eliminate it from the manuscript. For option 1, you need to go beyond what was done in Lorenzi et al. (2016) regarding protein features, potential functional domains and potential homologues in other species. There are more sophisticated and sensitive methods that were not used here, such as HMM profiles-based searches, searching for deep homologues using structural predictions, etc.

Response: We opted to make it more concise. We are interested in the incorporation of FamB into the ST multigenic families. Besides that FamB and FamC genes encode proteins with low conservation among themselves and that there are no structural identities or conserved domains and motifs. In this version we emphasize the systems we use to analyze structurally similar domains and genes in the text (lines 227 to 231). We also incorporate a study carried out in ProtVirDB

2.6: As conceived, this sequence analysis is not informative. To be aligned together, Fams A, B and C should be known to be homologous (i.e., have some level of sequence similarity. Aligning unrelated sequences doesn’t make sense, and any phylogenetic reconstruction would be uninformative. Even if they were related, a tree with members of the 3 fams and no outgroups is by definition unrooted, so that no inferences about “what came first” can be made.

Response: We agree with the comment. The phylogenetic tree was removed. Sections 2.5 and 2.6 were joined into 2.5

 

It may be worth doing an alignment between FamB and C if they share some similarity. If the authors want to include these analysis, I’d like to see a list of accession numbers for the sequences employed, an alignment figure as supplementary data and details of how the tree/s was/were inferred. For protein sequence alignment I prefer using a more modern program, such as MUSCLE or MAFFT instead of Clustal.

Response: The alignment was done by Muscle and the analysis was incorporated into figure 5.

 

 

2.7: Gene family size results from a combination of gene gains and loses, and it is very hard to determine the exact course of events along a phylogeny, especially when the number of genes and species is low, as in this case. I think it’s safe to suggest (although not to conclude) that FamB and FamC expanded in the lineage leading to T. gondii, but one shouldn’t say the same for H. hammondi. I’m not sure that figure 5 is worth showing in the manuscript. Same for figure 7B.

Response: as mentioned, we limit the concept of gene expansion only for T. gondii, as a possibility. We decided to remove Figure 5 and 7b and Fig. S3.

 

Line 626: the word ‘idea’ is incorrectly used (I see that it translates like that from the mode it’s used in another language). It should be “a more detailed picture of…”

Response:

Since we removed this paragraph from the discussion, the wrong word was also removed.

In this version the length of the STs Ia_R and XII_R were corrected (Table 1 and S1). The average length of the STs, was also corrected. Given the overall changes made, the order of the tables was reordered.

Based on the new additions in results, general aspects of the discussion were modified (see changes in red font).

Round 2

Reviewer 2 Report

The authors have addressed my concerns and request satisfactorily. Thank you.