Horizontally transferred cell-free chromatin particles function as autonomous “predatory” genomes and vehicles for transposable elements within host cells

Reviewer #1 (Public review):

Summary:

Horizontal gene transfer is the transmission of genetic material between organisms through ways other than reproduction. Frequent in prokaryotes, this mode of genetic exchange is scarcer in eukaryotes, especially in multicellular eukaryotes. Furthermore, the mechanisms involved in eukaryotic HGT are unknown. This article by Banerjee et al. claims that HGT occurs massively between cells of multicellular organisms. According to this study, the cell free chromatin particles (cfChPs) that are massively released by dying cells are incorporated in the nucleus of neighboring cells. These cfChPs are frequently rearranged and amplified to form concatemers, they are made of open chromatin, expressed, and capable of producing proteins. Furthermore, the study also suggests that cfChPs transmit transposable elements (TEs) between cells on a regular basis, and that these TEs can transpose, multiply, and invade receiving cells. These conclusions are based on a series of experiments consisting in releasing cfChPs isolated from various human sera into the culture medium of mouse cells, and using FISH and immunofluorescence to monitor the state and fate of cfChPs after several passages of the mouse cell line.

Strengths:

The results presented in this study are interesting because they may reveal unsuspected properties of some cell types that may be able to internalize free-circulating chromatin, leading to its chromosomal incorporation, expression, and unleashing of TEs. The authors propose that this phenomenon may have profound impacts in terms of diseases and genome evolution. They even suggest that this could occur in germ cells, leading to within-organism HGT with long-term consequences.

Weaknesses:

The claims of massive HGT between cells through internalization of cfChPs are not well supported because they are only based on evidence from one type of methodological approach: immunofluorescence and fluorescent in situ hybridization (FISH) using protein antibodies and DNA probes. Yet, such strong claims require validation by at least one, but preferably multiple, additional orthogonal approaches. This includes, for example, whole genome sequencing (to validate concatemerization, integration in receiving cells, transposition in receiving cells), RNA-seq (to validate expression), ChiP-seq (to validate chromatin state).

Another weakness of this study is that it is performed only in one receiving cell type (NIH3T3 mouse cells). Thus, rather than a general phenomenon occurring on a massive scale in every multicellular organism, it could merely reflect aberrant properties of a cell line that for some reason became permeable to exogenous cfChPs. This begs the question of the relevance of this study for living organisms.

Should HGT through internalization of circulating chromatin occur on a massive scale, as claimed in this study, and as illustrated by the many FISH foci observed in Fig 3 for example, one would expect that the level of somatic mosaicism may be so high that it would prevent assembling a contiguous genome for a given organism. Yet, telomere-to-telomere genomes have been produced for many eukaryote species, calling into question the conclusions of this study.

Reviewer #2 (Public review):

I must note that my comments pertain to the evolutionary interpretations rather than the study's technical results. The techniques appear to be appropriately applied and interpreted, but I do not feel sufficiently qualified to assess this aspect of the work in detail.

I was repeatedly puzzled by the use of the term "function." Part of the issue may stem from slightly different interpretations of this word in different fields. In my understanding, "function" should denote not just what a structure does, but what it has been selected for. In this context, where it is unclear if cfChPs have been selected for in any way, the use of this term seems questionable.

Similarly, the term "predatory genome," used in the title and throughout the paper, appears ambiguous and unjustified. At this stage, I am unconvinced that cfChPs provide any evolutionary advantage to the genome. It is entirely possible that these structures have no function whatsoever and could simply be byproducts of other processes. The findings presented in this study do not rule out this neutral hypothesis. Alternatively, some particular components of the genome could be driving the process and may have been selected to do so. This brings us to the hypothesis that cfChPs could serve as vehicles for transposable elements. While speculative, this idea seems to be compatible with the study's findings and merits further exploration.

I also found some elements of the discussion unclear and speculative, particularly the final section on the evolution of mammals. If the intention is simply to highlight the evolutionary impact of horizontal transfer of transposable elements (e.g., as a source of new mutations), this should be explicitly stated. In any case, this part of the discussion requires further clarification and justification.

In summary, this study presents important new findings on the behavior of cfChPs when introduced into a foreign cellular context. However, it overextends its evolutionary interpretations, often in an unclear and speculative manner. The concept of the "predatory genome" should be better defined and justified or removed altogether. Conversely, the suggestion that cfChPs may function at the level of transposable elements (rather than the entire genome or organism) could be given more emphasis.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

Horizontal gene transfer is the transmission of genetic material between organisms through ways other than reproduction. Frequent in prokaryotes, this mode of genetic exchange is scarcer in eukaryotes, especially in multicellular eukaryotes. Furthermore, the mechanisms involved in eukaryotic HGT are unknown. This article by Banerjee et al. claims that HGT occurs massively between cells of multicellular organisms. According to this study, the cell free chromatin particles (cfChPs) that are massively released by dying cells are incorporated in the nucleus of neighboring cells. These cfChPs are frequently rearranged and amplified to form concatemers, they are made of open chromatin, expressed, and capable of producing proteins. Furthermore, the study also suggests that cfChPs transmit transposable elements (TEs) between cells on a regular basis, and that these TEs can transpose, multiply, and invade receiving cells. These conclusions are based on a series of experiments consisting in releasing cfChPs isolated from various human sera into the culture medium of mouse cells, and using FISH and immunofluorescence to monitor the state and fate of cfChPs after several passages of the mouse cell line.

Strengths:

The results presented in this study are interesting because they may reveal unsuspected properties of some cell types that may be able to internalize free-circulating chromatin, leading to its chromosomal incorporation, expression, and unleashing of TEs. The authors propose that this phenomenon may have profound impacts in terms of diseases and genome evolution. They even suggest that this could occur in germ cells, leading to within-organism HGT with long-term consequences.

Weaknesses:

The claims of massive HGT between cells through internalization of cfChPs are not well supported because they are only based on evidence from one type of methodological approach: immunofluorescence and fluorescent in situ hybridization (FISH) using protein antibodies and DNA probes. Yet, such strong claims require validation by at least one, but preferably multiple, additional orthogonal approaches. This includes, for example, whole genome sequencing (to validate concatemerization, integration in receiving cells, transposition in receiving cells), RNA-seq (to validate expression), ChiP-seq (to validate chromatin state).

We agree with the reviewer’s suggestions. We propose to use RNA-seq using an orthogonal platform as a solution. This will allow us to answer multiple questions viz. validation of expression of human DNA in mouse cells, obtaining a detailed insight into genes and pathways driven by human cfChPs and enable us to identify chimeric human and mouse transcripts.

Another weakness of this study is that it is performed only in one receiving cell type (NIH3T3 mouse cells). Thus, rather than a general phenomenon occurring on a massive scale in every multicellular organism, it could merely reflect aberrant properties of a cell line that for some reason became permeable to exogenous cfChPs. This begs the question of the relevance of this study for living organisms.

We agree with the reviewer’s suggestion. We propose to show horizontal transfer of cfChPs using four different cell-lines representing four different species.

Should HGT through internalization of circulating chromatin occur on a massive scale, as claimed in this study, and as illustrated by the many FISH foci observed in Fig 3 for example, one would expect that the level of somatic mosaicism may be so high that it would prevent assembling a contiguous genome for a given organism. Yet, telomere-to-telomere genomes have been produced for many eukaryote species, calling into question the conclusions of this study.

The reviewer is right in expecting that the level of somatic mosaicism may be so high that it would prevent assembling a contiguous genome. This is indeed the case, and we find that beyond ~ 250 passages the genomes of the cfChPs treated NIH3T3 cells begin to die out apparently become their genomes have become too unstable for survival. This point will be highlighted in the revised version. It is likely that cell death resulting from large scale HGT creates a vicious cycle of more cell death induced by cfChPs thereby helping to explain the massive daily turnover of cells in the body (10⁹ – 10¹² cells per day).

Reviewer #2 (Public review):

I must note that my comments pertain to the evolutionary interpretations rather than the study's technical results. The techniques appear to be appropriately applied and interpreted, but I do not feel sufficiently qualified to assess this aspect of the work in detail.

I was repeatedly puzzled by the use of the term "function." Part of the issue may stem from slightly different interpretations of this word in different fields. In my understanding, "function" should denote not just what a structure does, but what it has been selected for. In this context, where it is unclear if cfChPs have been selected for in any way, the use of this term seems questionable.

We think this is a matter of semantics. We have used the term “function” since cfChPs that enter the cell are biologically active; they transcribe, translate, synthesize, proteins and proliferate. We, therefore feel that the term function is not inappropriate.

Similarly, the term "predatory genome," used in the title and throughout the paper, appears ambiguous and unjustified. At this stage, I am unconvinced that cfChPs provide any evolutionary advantage to the genome. It is entirely possible that these structures have no function whatsoever and could simply be byproducts of other processes. The findings presented in this study do not rule out this neutral hypothesis. Alternatively, some particular components of the genome could be driving the process and may have been selected to do so. This brings us to the hypothesis that cfChPs could serve as vehicles for transposable elements. While speculative, this idea seems to be compatible with the study's findings and merits further exploration.

We take the reviewer’s point. We will replace the term “predatory genome” with a more neutral and factual term “supernumerary genome” in the title and throughout the manuscript in the revised version.

I also found some elements of the discussion unclear and speculative, particularly the final section on the evolution of mammals. If the intention is simply to highlight the evolutionary impact of horizontal transfer of transposable elements (e.g., as a source of new mutations), this should be explicitly stated. In any case, this part of the discussion requires further clarification and justification.

We propose to revise the “discussion” section taking into account the issues raised by the reviewer and highlight the potential role of cfChPs in evolution by acting as vehicles of transposable elements.

In summary, this study presents important new findings on the behavior of cfChPs when introduced into a foreign cellular context. However, it overextends its evolutionary interpretations, often in an unclear and speculative manner. The concept of the "predatory genome" should be better defined and justified or removed altogether. Conversely, the suggestion that cfChPs may function at the level of transposable elements (rather than the entire genome or organism) could be given more emphasis.

Our responses to this paragraph are given in the two above sections.