The GC-content at the 5’ends of human protein-coding genes is undergoing mutational decay

  1. Yi Qiu
  2. Yoon Mo Kang
  3. Christopher Korfmann
  4. Fanny Pouyet
  5. Andrew Eckford
  6. Alexander F. Palazzo

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Abstract

In vertebrates, most protein-coding genes have a peak of GC-content near their 5’ transcriptional start site (TSS). This feature promotes both the efficient nuclear export and translation of mRNAs. Despite the importance of GC-content for RNA metabolism, its general features, origin, and maintenance remain mysterious. We investigated the evolutionary forces shaping GC-content at the transcriptional start site (TSS) of genes through both comparative genomic analysis of nucleotide substitution rates between different species and by examining human de novo mutations. Our data suggests that GC-peaks at TSSs were present in the last vertebrate common ancestor and are largely dictated by recombination patterns. We observe that in primates and rodents, where recombination is directed away from TSSs by PRDM9, GC-content at protein-coding gene TSSs is currently undergoing mutational decay. In canids, which lack PRDM9 and perform recombination at TSSs, GC-content at protein-coding gene TSSs is increasing. These patterns extend into the open reading frame affecting protein-coding regions, and we show that changes in GC-content due to recombination affect synonymous codon position choices at the start of the open reading frame. Our results indicate that although high GC-content in protein-coding genes may be shaped by selective pressures to enhance expression, the dynamics of GC-content in mammals are largely shaped by patterns of recombination.

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    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    This manuscript investigates the dynamics of GC-content patterns in the 5'end of the transcription start sites (TSS) of protein-coding genes (pc-genes). The manuscript introduces a quite careful and comprehensive analysis of GC content in pc-genes in humans and other vertebrates, specially around the TSS. The result of this investigation states that "GC-content surrounding the TSS is largely influenced by patterns of recombination." (from end of Introduction)

    My main concern with this manuscript is one of causal reasoning, whether intended or not. I hope the authors can follow my reasoning bellow on how the logic sometimes seems to fail, and that they introduce changes to clarify their suggested mechanisms of action.

    The above quoted sentence form the end of the Intro is in conflict with this other sentence that appears at the end of the Abstract "the dynamics of GC-content in mammals are largely shaped by patterns of recombination". The sentence in the Intro seems to indicate that the effect is specific to TSSs, but the one in the abstract seem to indicate the opposite, that is, that the effect is ubiquitous.

    We are sorry about the lack of clarity. We have now rewritten the abstract and intro to emphasize that our results are restricted to the 5' end of genes, and that by "patterns of recombination" we mean "historic patterns of recombination".

    The observations as stated in the abstract are: "We observe that in primates and rodents, where recombination is directed away from TSSs by PRDM9, GC-content at protein-coding gene TSSs is currently undergoing mutational decay."

    If I understand the measurements described in the manuscript correctly, and the arguments around them, you seem to show that the mutational decay of GC-content in humans is independent of location (TSSS or not), as noted here (also from the abstract) "These patterns extend into the open reading frame affecting protein-coding regions, and we show that changes in GC-content due to recombination affect synonymous codon position choices at the start of the open reading frame."

    Again, we have rewritten this section to clarify these points.

    There is one more result described in the manuscript, that in my mind is very important, but it is not given the relevance that it appears to me that it has. That is presented in Figure S3G. "we concluded that GC-content at the TSS of protein-coding genes is not at equilibrium, but in decay in primates and rodents. This decay rate is similar to the decay seen in intergenic regions that have the same GC-content (Figure S3G)"

    Thus, if the decaying effect happens everywhere, how can it be related to "recombination being directed away from TSSs by PRDM9" as it is stated in the abstract and in the model described in Figure 7?

    We make the argument that the GC-peak as likely caused by past recombination events. This is based on:

    1. The change in GC-content at the TSS in Dogs and Fox, coupled to the fact that they perform recombination at the TSS
    1. That the TSS can act as a default recombination site in mice when PRDM9 is knocked out
    1. That some forms of PRDM9 allow for recombination at TSS (see Schield et al., 2020, Hoge et al. 2023, and Joseph et al., 2023) and that this is expected to cause an increase in GC-content

    We thus speculate that the GC-peak in humans and rodents was caused by past recombination at TSSs that were permitted by ancient variants of PRDM9. We further point out that PRDM9 is undergoing rapid evolution, and some of the past versions of the protein may have had this property.

    We have tried to clarify these points in the latest version of the text.

    The fact that the decay rate is similar to any other region with similar GC-content should be an indication that the effect is not related to anything having to do with TSS or recombination being directed away from TSSs by PRDM9.

    We are sorry about the lack of clarity. TSSs in humans, chimpanzees, mouse and rats are are experiencing GC-decay at the same rate as in non-functional DNA regions with high GC-content. Thus the GC-peak is not being maintained by selection. This is surprising, given the role that GC-content plays in gene expression. This is a critical point, and we added it to the "conclusion" section of the abstract.

    I hope these paragraphs show my confusion about the relationship between the results presented which I think are very comprehensive and their interpretation and suggested model for GC-content dynamics around TSSs in human.

    On another note, can you provided a bit more background on recombination and its mechanisms?

    We have done our best to clarify these issues.

    You seem to have confident sets of genes under high/low/med recombination. How are those determined.

    We used the recombination rates per gene provided in Pouyet et al 2017 to identify the sets of genes under low/med/high recombination. Those rates were estimated from the HapMap genetic map (Frazer et al., 2007). This is now all specified in the methods section.

    You also seem to concentrate the cause of recombination on PRDM9, please explain. Is PRDM9 the unique indicator of recombination?

    PRDM9 has been shown to be the primary determinant of where recombination occurs in the genome (Grey et al., 2011, Brick et al., 2012). This is very well established. We now reword some of the introduction to make this clear.

    specific comments

    Figure 1, it is very hard to understand the differences between the three rows. Please explain more clearly in the legend, and add more information to the figure itself.

    We altered the axis titles to make this clearer. We also label "Upsream", "Exon 1" and "Part of Intron 1" in Figure 1C, F and I, and in Figure 2C. We now spell this out in the Figure Legend.

    Figure 7, express somewhere in the figure that the y axis measures GC content.

    We now added "GC Content" to the left of the first "graph" in Figure 7.

    Figure seems to introduce a 'causal' model of GC-content dismissing (diminishing?) based on recombination being directed away from TSSs. How about the diminishing of GC-content on any other genomic regions as you have shown in Figure S3G?

    Our focus in this model, and manuscript, is on TSSs. I think that to add the dynamics of other GC-rich regions is distracting. We do not know what caused these intergenic genomic regions to be high in GC-content prior to decay. After excluding known recombination sites and TSSs, these regions are very rare in the human genome. They may be ancient recombination sites that are decaying in GC-content. However, unlike TSSs, which have some connection to recombination (i.e. data from PRDM9 knockout mice and dogs and fox), we do not have any direct or indirect evidence that these other sites were used for recombination in the past. Alternatively, there could have been some other pressure on these sites in the past to increase GC-content that we are not aware of.

    -- The title is too selective, as to the results, and it has the implication that the decay is exclusive to the surrounding of the TSSs.

    Decay of GC-content towards equilibrium is the default state for non-functional DNA. That it is occurring at the TSS is surprising, as it indicates that the GC-peak is not maintained by selection. We now state this in the paper and include this in the "conclusion" portion of the abstract.

    Reviewer #1 (Significance (Required)):

    The statistical analysis is comprehensive and robust.

    We thank the reviewer for this.

    Their model interpretation as is describe induces confusion and needs to be clarified.

    We are sorry about this. Hopefully our revised text will clear up the confusion.

    I am an expert computational biologist, I do not have a deep knowledge of sequence implications of recombination, and it would be good if the manuscript could add some more background on that.

    We thank the reviewer for their perspective, and we hope that our text changes better explain to the non-expert why our findings are so surprising. We further clarify how recombination affects DNA sequence by gBGC and some of these changes are detailed in our response to the other reviewers.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    In this work, the author present various analyses suggesting that GC-content in TSS of coding genes is affected by recombination. The article findings are interesting and novel and are important to our understanding of how various non-adaptive evolutionary forces shape vertebrate genome evolutionary history.

    We thank the reviewer for these kind words.

    The Methods section includes most needed details (see comments below for missing information), and the scripts and data provided online help in transparency and usability of these analyses.

    I have several comments, mostly regarding clarifications in the text and several suggestions:

    1. In introduction: CpG islands, have been shown to activate transcription (Fenouil et al., 2012) - what is known about CpG Islands is somewhat inaccurately described. It should be rephrased more accurately, e.g. - CpG Islands found near TSS are associated with robust and high expression level of genes, including genes expressed in many tissues, such as housekeeping genes.

    We thank the reviewer for that. We have rewrote this part of the introduction.

    1. The following claim (in Introduction), regarding retrogenes and their GC content is not in agreement recent analyses: "Indeed, it has been observed that these genes have elevated GC-content at their 5' ends in comparison to their intron-containing counterparts, suggesting that elevation of GC-content can be driven by positive selection to drive their efficient export (Mordstein et al., 2020). Moreover, retrogenes tend to arise from parental genes that have high GC-content at their 5'ends (Kaessmann et al.,2009)." Recent work showed that retrogenes in mouse and human are significantly depleted of CpG islands in their promoters (PMID: 37055747). This follows the notion that young genes, such as these retrogenes, have simple promoters (PMID: 30395322) with few TF binding sites and without CpGs. The two reported trends should be both mentioned with some suggestions regarding why they seem to be contrasting each other and how they can be reconciled.

    We thank the reviewer for this information. The previous report (Mordstein et al., 2020) indicated that the increase in GC-content occurs downstream of the TSS in retrogenes. Since sequences upstream of the TSS are not part of the retro-insertion, it is not surprising that GC-content may differ between the retrogene and the parental gene. That retrogenes have lower numbers of CpGs upstream of the TSS, bolsters the idea that GC-content is not required for transcription and that the GC-peak is not being maintained in most genes by purging selection.

    1. In "Thus GC-content is expected, and is indeed observed to be higher near recombination hotspots due to gBGC (REF)." I think you forgot the reference...

    We thank the reviewer for catching this.

    1. In Results, regarding average GC content (Fig 2X): "Interestingly, this pattern is different in the nonamniotes examined, including anole lizard, coelacanth, shark and lamprey." - in lizard, it seems that the genomic average is lower (and lizards are amniotes)

    You are absolutely right. We now fix this.

    1. In Discussion, the statement: "This model is supported by findings in a recent preprint, which documents the equilibrium state of GC-content in TSS regions from numerous organisms" seems to contrast with the findings of the mentioned preprint. If "most mammals have a high GC-content equilibrium state" but still have a functional PRDM9, in the lack of evidence for functional differences between ortholog PRDM9 proteins (such as signatures for positive selection or functional assays), the authors' findings regarding the relationship between a lack of PRDM9 in canids and the trends observed in their TSS, are weakened.

    We are sorry about the confusion. We were not exactly sure what points were being commented on. 1) whether GC-content is at equilibrium for most mammals or 2) that the equilibrium state is high for most mammals despite containing PRDM9. We rewrote this sentence to clarify both issues (especially given that these concepts may not be clear to non-experts, such as the first reviewer). To answer the first potential concern, the paper in question (Joseph et al., 2023), does not show that GC-content at the TSS in mammals is at equilibrium, rather, it calculates what the equilibrium state is given the nucleotide substitution rates. In most organisms, the TSS is not at equilibrium. To answer both 1 and 2, Joseph et al., show that the equilibrium GC-content at the TSS for canids is much higher than for other mammals. They and others infer that the diversity between other mammals (where the equilibrium state is higher than humans and rodents but lower than canids) has to do with the variation between PRDM9 orthologues, however this has yet to be tested. Although the action of PRDM9 has not been evaluated in most mammals, we do point out that in snakes PRDM9 allows for some recombination at the TSS.

    1. In Methods, the ENSEMBL version (in addition of the per-species genome version) should be mentioned.

    This has been fixed.

    1. In Fig 1, it is worth clarifying in the legend that the differences between the first and second rows of panels is in the length of the plotted region.

    We have now indicated this in the figure legend.

    Reviewer #2 (Significance (Required)):

    The manuscript provides a rigorous analysis of the possible processes that have impacted the TSS GC-content during evolution. It should be of interest to a diverse set of investigators in the genomics community, since it touches on different topics including genome evolution, transcription and gene structures.

    Thank you.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    This study analyzes the distribution of GC-content along genes in humans and vertebrates, and particularly the higher GC-content in the 5'-end than in the 3'-end of genes. The results suggest that this pattern is ancient in vertebrates, currently decaying in mouse and humans, and probably driven by recombination and GC-biased gene conversion. It is proposed that the 5'-3' gradient was generated during evolution when PRDM9 was less active (in which case recombination occurs mostly near transcription start sites), and decays when PRDM9 is very active, as it is currently in humans and mouse. This is a very interesting hypothesis, also corroborated by a recent, similar analysis in mammals (Joseph et al. 2023). These two preprints, which appeared around the same time, are, I think, quite novel and important. The analyses performed here are thorough and convincing. Source code and raw data sets are openly distributed. I only have a couple of minor comments and suggestions, which I hope might help improve the manuscript.

    Thank you very much for the kind words.

    A1. There has been quite some work on the 5'-3' GC-content gradient in plants (e.g. Clément et al. 2014 GBE, Ressayre et al. 2015 GBE, Brazier & Glemin 2023 biorxiv), which you might like to cite.

    Thank you for pointing out these very interesting papers, we have incorporated them into the latest version.

    A2. CpG-content and GC-content are related in various ways (e.g. see Galtier & Duret 2000 MBE, Fryxell & Moon 2005 MBE) that you might like to discuss; currently the manuscript discusses the CpG hypermutation rate as a driver of GC-content but the picture might be a bit more complex.

    Thank you for this, we have incorporated these citations.

    A3. The model introduced by this manuscript (figure 7) is dependent on the evolution of recombination determination in vertebrates and the role of PRDM9. A recent preprint by Raynaud et al (biorxiv) seems relevant to this issue.

    Thank you for pointing out this pre-print. We have added a paragraph to the discussion that mentions this work. This also initiated a conversation with the authors, and we include some "personal communications" that illuminate what is going on in teleost fish.

    Line-by-line comments

    B1. "First, highly spliced mRNAs tend to have high GC-content at their 5' ends despite the fact that it is not required for export and does not affect expression levels (Mordstein et al., 2020)" -> I do not totally understand this sentence, which seems to imply some link between splicing and export/expression, could you please clarify?

    We rewrote that sentence to make it clearer.

    B2. "mismatches will form in the heteroduplex which are typically corrected in favor of Gs and Cs over As and Ts by about 70%" -> This 70% figure is human-specific, and varies a lot among species; I know in this introduction you're mainly reviewing the human literature but since this part of the text introduces gBGC as a process maybe clarify by adding "in humans" or refrain from giving this figure?

    Thank you. This is a good point. We fixed this.

    B3. "Thus GC-content is expected, and is indeed observed to be higher near recombination hotspots due to gBGC (REF)." -> reference missing here; actually I'm not sure you will find a good reference for this because PRDM9-dependent hotspots are so short-lived that GC-content would only respond weakly; mayber rather refer to the equilibrium GC-content (and cite, for instance, Pratto et al 2014 Science), or to high-recombining regions instead of hotspots (and you have plenty of papers to cite)?

    Thanks for this.

    B4. Paragraph starting: "PRDM9 and recombination hotspots also experience accelerated rates of evolution..." -> I would suggest removing the word "also" and moving this paragraph up, just before the sentence I'm commenting above (the one starting "Thus GC-content..."). This will justify my suggestion in comment B3 of mentioning high-recombining regions instead of hotspots, while also avoiding to have the important paragraph on recombination at TSS (the one starting "There are interesting connections...") being sandwiched between two sections on PRDM9.

    We did not move this paragraph, although we did adjust the wording slightly.

    B5. Paragraph starting "There are interesting connections..." is crucial to your discussion and might be emphasized a bit more in introduction, in my opinion. For instance, what about adding a sentence like "Also not directly relevant to humans, these observations suggest that gBGC might have played a role in shaping the observed 5'-3' GC-content gradient."

    We did not alter the structure of this paragraph but we did reword sections of it.

    1. "Interestingly, this pattern is different in the non-amniotes examined, including anole lizard, coelacanth, shark and lamprey. These organisms had clear differences in GC-content between their first exon and surrounding sequences (upstream and intronic sequences), which came close to the overall genomic GC-content." -> I'm not sure I got the point the authors are intending to make here. Also please note that lizards are amniotes.

    We thank the reviewer for catching this error, we have fixed this.

    Reviewer #3 (Significance (Required)):

    This is one of two preprints having appeared ~at the same time (the other one being the cited Joseph et al 2023), which I think are quite important and convincing regarding the role of PRDM9-dependent and PRDM9-independent recombination on GC-content evolution in vertebrates. I support publication of this preprint in a molecular evolutionary journal.

    We thank the reviewer for their kind assessment!

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    Referee #3

    Evidence, reproducibility and clarity

    This study analyzes the distribution of GC-content along genes in humans and vertebrates, and particularly the higher GC-content in the 5'-end than in the 3'-end of genes. The results suggest that this pattern is ancient in vertebrates, currently decaying in mouse and humans, and probably driven by recombination and GC-biased gene conversion. It is proposed that the 5'-3' gradient hass generated during evolution when PRDM9 was less active (in which case recombination occurs mostly near transcription start sites), and decays when PRDM9 is very active, as it is currently in humans and mouse. This is a very interesting hypothesis, also corroborated by a recent, similar analysis in mammals (Joseph et al. 2023). These two preprints, which appeared around the same time, are, I think, quite novel and important. The analyses performed here are thorough and convincing. Source code and raw data sets are openly distributed. I only have a couple of minor comments and suggestions, which I hope might help improve the manuscript.

    A1. There has been quite some work on the 5'-3' GC-content gradient in plants (e.g. Clément et al. 2014 GBE, Ressayre et al. 2015 GBE, Brazier & Glemin 2023 biorxiv), which you might like to cite.

    A2. CpG-content and GC-content are related in various ways (e.g. see Galtier & Duret 2000 MBE, Fryxell & Moon 2005 MBE) that you might like to discuss; currently the manuscript discusses the CpG hypermutation rate as a driver of GC-content but the picture might be a bit more complex.

    A3. The model introduced by this manuscript (figure 7) is dependent on the evolution of recombination determination in vertebrates and the role of PRDM9. A recent preprint by Raynaud et al (biorxiv) seems relevant to this issue.

    Line-by-line comments

    B1. "First, highly spliced mRNAs tend to have high GC-content at their 5' ends despite the fact that it is not required for export and does not affect expression levels (Mordstein et al., 2020)" -> I do not totally understand this sentence, which seems to imply some link between splicing and export/expression, could you please clarify?

    B2. "mismatches will form in the heteroduplex which are typically corrected in favor of Gs and Cs over As and Ts by about 70%" -> This 70% figure is human-specific, and varies a lot among species; I know in this introduction you're mainly reviewing the human literature but since since this part of the text introduces gBGC as a process maybe clarify by adding "in humans" or refrain from giving this figure?

    B3. "Thus GC-content is expected, and is indeed observed to be higher near recombination hotspots due to gBGC (REF)." -> reference missing here; actually I'm not sure you will find a good reference for this because PRDM9-dependent hotspots are so short-lived that GC-content would only respond weakly; mayber rather refer to the equilibrium GC-content (and cite, for instance, Pratto et al 2014 Science), or to high-recombining regions instead of hotspots (and you have plenty of papers to cite)?

    B4. Paragraph starting: "PRDM9 and recombination hotspots also experience accelerated rates of evolution..." -> I would suggest removing the word "also" and moving this paragraph up, just before the sentence I'm commenting above (the one starting "Thus GC-content..."). This will justify my suggestion in comment B3 of mentioning high-recombining regions instead of hotspots, while also avoiding to have the important paragraph on recombination at TSS (the one starting "There are interesting connections...") being sandwiched between two sections on PRDM9.

    B5. Paragraph starting "There are interesting connections..." is crucial to your discussion and might be emphasized a bit more in introduction, in my opinion. For instance, what about adding a sentence like "Also not directly relevant to humans, these observations suggest that gBGC might have played a role in shaping the observed 5'-3' GC-content gradient."

    1. "Interestingly, this pattern is different in the non-amniotes examined, including anole lizard, coelacanth, shark and lamprey. These organisms had clear differences in GC-content between their first exon and surrounding sequences (upstream and intronic sequences), which came close to the overall genomic GC-content." -> I'm not sure I got the point the authors are intending to make here. Also please note that lizards are amniotes.

    Significance

    This is one of two preprints having appeared ~at the same time (the other one being the cited Joseph et al 2023), which I think are quite important and convincing regarding the role of PRDM9-dependent and PRDM9-independent recombination on GC-content evolution in vertebrates. I support publication of this preprint in a molecular evolutionary journal.

  3. Review Commons

    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

    In this work, the author present various analyses suggesting that GC-content in TSS of coding genes is affected by recombination. The article findings are interesting and novel and are important to our understanding of how various non-adaptive evolutionary forces shape vertebrate genome evolutionary history.

    The Methods section includes most needed details (see comments below for missing information), and the scripts and data provided online help in transparency and usability of these analyses.

    I have several comments, mostly regarding clarifications in the text and several suggestions:

    1. In introduction: CpG islands, have been shown to activate transcription (Fenouil et al., 2012) - what is known about CpG Islands is somewhat inaccurately described. It should be rephrased more accurately, e.g. - CpG Islands found near TSS are associated with robust and high expression level of genes, including genes expressed in many tissues, such as housekeeping genes.
    2. The following claim (in Introduction), regarding retrogenes and their GC content is not in agreement recent analyses: "Indeed, it has been observed that these genes have elevated GC-content at their 5' ends in comparison to their intron-containing counterparts, suggesting that elevation of GC-content can be driven by positive selection to drive their efficient export (Mordstein et al., 2020). Moreover, retrogenes tend to arise from parental genes that have high GC-content at their 5'ends (Kaessmann et al.,2009)." Recent work showed that retrogenes in mouse and human are significantly depleted of CpG islands in their promoters (PMID: 37055747). This follows the notion that young genes, such as these retrogenes, have simple promoters (PMID: 30395322) with few TF binding sites and without CpGs.
      The two reported trends should be both mentioned with some suggestions regarding why they seem to be contrasting each other and how they can be reconciled.
    3. In "Thus GC-content is expected, and is indeed observed to be higher near recombination hotspots due to gBGC (REF)." I think you forgot the reference...
    4. In Results, regarding average GC content (Fig 2X): "Interestingly, this pattern is different in the nonamniotes examined, including anole lizard, coelacanth, shark and lamprey." - in lizard, it seems that the genomic average is lower (and lizards are amniotes)
    5. In Discussion, the statement: "This model is supported by findings in a recent preprint, which documents the equilibrium state of GC-content in TSS regions from numerous organisms" seems to contrast with the findings of the mentioned preprint. If "most mammals have a high GC-content equilibrium state" but still have a functional PRDM9, in the lack of evidence for functional differences between ortholog PRDM9 proteins (such as signatures for positive selection or functional assays), the authors' findings regarding the relationship between a lack of PRDM9 in canids and the trends observed in their TSS, are weakened.
    6. In Methods, the ENSEMBL version (in addition of the per-species genome version) should be mentioned.
    7. In Fig 1, it is worth clarifying in the legend that the differences between the first and second rows of panels is in the length of the plotted region.

    Significance

    The manuscript provides a rigorous analysis of the possible processes that have impacted the TSS GC-content during evolution. It should be of interest to a diverse set of investigators in the genomics community, since it touches on different topics including genome evolution,transcription and gene structures.

  4. Review Commons

    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

    This manuscript investigates the dynamics of GC-content patterns in the 5'end of the transcription start sites (TSS) of protein-coding genes (pc-genes). The manuscript introduces a quite careful and comprehensive analysis of GC content in pc-genes in humans and other vertebrates, specially around the TSS. The result of this investigation states that "GC-content surrounding the TSS is largely influenced by patterns of recombination." (from end of Introduction)

    My main concern with this manuscript is one of causal reasoning, whether intended or not. I hope the authors can follow my reasoning bellow on how the logic sometimes seems to fail, and that they introduce changes to clarify their suggested mechanisms of action.

    The above quoted sentence form the end of the Intro is in conflict with this other sentence that appears at the end of the Abstract "the dynamics of GC-content in mammals are largely shaped by patterns of recombination". The sentence in the Intro seems to indicate that the effect is specific to TSSs, but the one in the abstract seem to indicate the opposite, that is, that the effect is ubiquitous.

    The observations as stated in the abstract are: "We observe that in primates and rodents, where recombination is directed away from TSSs by PRDM9, GC-content at protein-coding gene TSSs is currently undergoing mutational decay."

    If I understand the measurements described in the manuscript correctly, and the arguments around them, you seem to show that the mutational decay of GC-content in humans is independent of location (TSSS or not), as noted here

    (also from the abstract) "These patterns extend into the open reading frame affecting protein-coding regions, and we show that changes in GC-content due to recombination affect synonymous codon position choices at the start of the open reading frame."

    There is one more result described in the manuscript, that in my mind is very important, but it is not given the relevance that it appears to me that it has. That is presented in Figure S3G. "we concluded that GC-content at the TSS of protein-coding genes is not at equilibrium, but in decay in primates and rodents. This decay rate is similar to the decay seen in intergenic regions that have the same GC-content (Figure S3G)"

    Thus, if the decaying effect happens everywhere, how can it be related to "recombination being directed away from TSSs by PRDM9" as it is stated in the abstract and in the model described in Figure 7?

    The fact that the decay rate is similar to any other region with similar GC-content should be an indication that the effect is not related to anything having to do with TSS or recombination being directed away from TSSs by PRDM9.

    I hope these paragraphs show my confusion about the relationship between the results presented which I think are very comprehensive and their interpretation and suggested model for GC-content dynamics around TSSs in human.

    On another note, can you provided a bit more background on recombination and its mechanisms? You seem to have confident sets of genes under high/low/med recombination. How are those determined. You also seem to concentrate the cause of recombination on PRDM9, please explain. Is PRDM9 the unique indicator of recombination?

    Specific comments

    Figure 1, it is very hard to understand the differences between the three rows. Please explain more clearly in the legend, and add more information to the figure itself.

    Figure 7, express somewhere in the figure that the y axis measures GC content. Figure seems to introduce a 'causal' model of GC-content dismissing based on recombination being directed away from TSSs. How about the diminishing of GC-content on any other genomic regions as you have shown in Figure S3G?

    The title is too selective, as to the results, and it has the implication that the decay is exclusive to the surrounding of the TSSs.

    Significance

    The statistical analysis is comprehensive and robust. Their model interpretation as is describe induces confusion and needs to be clarified.

    I am an expert computational biologist, I do not have a deep knowledge of sequence implications of recombination, and it would be good if the manuscript could add some more background on that.

  5. Version published to 10.1101/2024.03.12.584636 on bioRxiv