Intracellular growth of Chlamydia trachomatis leads to global histone hypermethylation by impairing demethylation
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eLife Assessment
This is a valuable study of changes in host genome histone methylation and transcription changes associated with Chlamydia infection. The data presented are solid but further analysis would strengthen the authors overall conclusions.
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Abstract
Chlamydia trachomatis , an intracellular bacterium, highjacks metabolites from the host cell for its own proliferation. We provide evidence of global hypermethylation of the host proteome, including histones, during the late stages of infection. Single cell analyses revealed co-occurrence of several methylated residues on histones. Histone hypermethylation correlated with bacterial load and was prevented by antibiotic treatment. Mapping of trimethylation of histone 3 at residues K4 and K9 revealed a broad distribution throughout the chromatin. Nuclear fractions of infected cells exhibited a four-fold decrease of demethylase activity towards H3K4me3 and a two-fold increase in succinate concentration, a competitive inhibitor for the demethylase co-factor α-ketoglutarate. Supplementation of the culture medium with dimethyl-ketoglutarate (DMKG) or with iron, a second co-factor of histone lysine demethylases, reduced histone hypermethylation. DMKG supplementation modified the transcription of about one third of the infection-responsive genes, indicating that histone hypermethylation contributes to modulating the transcriptional response of the host to infection. Finally, chemical inhibition of histone demethylases in a mouse model of infection showed a moderate benefit regarding the outcome of infection. Overall, our data show that the metabolic pressure exerted by a pathogen with an intracellular lifestyle drives epigenetic changes in infected cells.
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eLife Assessment
This is a valuable study of changes in host genome histone methylation and transcription changes associated with Chlamydia infection. The data presented are solid but further analysis would strengthen the authors overall conclusions.
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Reviewer #1 (Public Review):
This study by Charendoff et al provides interesting observations related to global histone hypermethylation in host cells, during Chlamydia trachomatis infections. The core observation they report is that the host histones are highly hypermethylated during infection, and this appears to be an amplifying effect due to continuous inhibition of demethylases, in part due to a metabolic shift in the host where succinate amounts (which inhibit demethylases) increases. The authors claim specifically due to the bacteria, since antibiotic treatment prevents histone hypermethylation (but leaves you wondering about cause/consequence correlations).
The core observation of hyper methylation is very interesting, and well documented. There are a number of points to consider though in order to fully substantiate the …
Reviewer #1 (Public Review):
This study by Charendoff et al provides interesting observations related to global histone hypermethylation in host cells, during Chlamydia trachomatis infections. The core observation they report is that the host histones are highly hypermethylated during infection, and this appears to be an amplifying effect due to continuous inhibition of demethylases, in part due to a metabolic shift in the host where succinate amounts (which inhibit demethylases) increases. The authors claim specifically due to the bacteria, since antibiotic treatment prevents histone hypermethylation (but leaves you wondering about cause/consequence correlations).
The core observation of hyper methylation is very interesting, and well documented. There are a number of points to consider though in order to fully substantiate the findings, and close out loose ends. My comments are broad - and built around the interpretations (vs the data presented).
(1) Related to observations coming Fig 1C etc, and connecting to Fig 3 - the hyper methylation appears to be across different protein arg/lys residues - and is not histone specific. So, is it just a consequence of high SAM pools and flux in infected cells? i.e. the bacterial infection increases SAM pools in cells, and provides an increase in substrate pools for the methyltransferases, leading to protein hyper methylation. The approach used here only measures steady-state SAM amounts (and not SAM flux or utilisation). For example, reduced SAM amounts in nuclei could be due to increased utilisation of SAM. The experiments done with the demethylase does not actually answer this question - if you decrease demethylase activity, you will get an increase in net methylation. The authors see an increase in net methylation in the infected cells - this would suggest that in addition (or perhaps primarily) to reduced demethylase activity, there could be much higher SAM utilisation/flux. Again, the over expression of JMJ proteins does not resolve this problem.
(2) Adding to this - what happens to SAM pools in the cells treated with the inhibitors? This actually may not look like the slightly reduced SAM pool observed in infected cell nuclei. Also, what is the SAM/SAH ratio (a very useful indicator of methylation activity).
(3) There is a correlation/implication issue here in Fig 2 - cells with C. trachoma's infection show hyper methylation. But these are the only cells with high C. trachomatis. So it is a bit ingenious to say that histone hyper methylation correlates with bacterial proliferation. The cells without bacteria don't have hyper methylation - and that does not have anything to do with the bacterial proliferation.
(4) The claim that demethylase activity is down in infected cells again comes primarily from the increased succinate (2-fold) amounts in infected nuclei - and then correlated with experiments where succinate, (permeable) a-KG are supplemented in excess. While I personally like the hypothesis that the hypermethylation might be a result of an imbalance in cofactors (succinate vs a-KG) in infected cells, the data presented is very premature to make that conclusion. Again, steady state measurements of only succinate cannot provide a clear answer to that question. For example, is there a clear allocation/flux difference (between a-KG, and leading out to glutamate/glutamine, vs flux through the TCA and increased succinate accumulation? Is there a bottleneck/build-up of succinate in cells that might lead to the increase in nuclei? This also opens another direction of possible regulation - increased histone succinylation. When you see a large increase in succinate in the nucleus, before looking at demethylase activity - it becomes obvious if succinate itself increases histone succinylation (through HATs).
(5) What might the authors hypothesise about why this hyper methylation happens? It appears in some ways that hyper methylation happens - potentially due to a metabolic bottleneck that the bacteria triggers (and there is a build-up of SAM and/or succinate, and altered flux out of a-kg). The methylation is just a visible outcome - but may not be central to pathogenesis or viability.
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Reviewer #2 (Public Review):
Strengths:
(1) Because the study compares genuinely infected cells with uninfected cells within the same infected cell population, it enables a clearer and more rigorous comparison.
(2) By using multiple Chlamydia species and cells from multiple host species (human and mouse), and obtaining consistent findings across these systems, the study demonstrates the generality of bacterium-induced epigenomic alterations.
(3) The study shows that the epigenomic changes are caused by reduced activity of JMJC domain-containing lysine demethylases, demonstrating through multiple complementary approaches-including the use of a demethylase inhibitor, overexpression of target-specific demethylases, and analysis from the perspective of cofactors required for JMJC domain-containing demethylases-that decreased lysine …
Reviewer #2 (Public Review):
Strengths:
(1) Because the study compares genuinely infected cells with uninfected cells within the same infected cell population, it enables a clearer and more rigorous comparison.
(2) By using multiple Chlamydia species and cells from multiple host species (human and mouse), and obtaining consistent findings across these systems, the study demonstrates the generality of bacterium-induced epigenomic alterations.
(3) The study shows that the epigenomic changes are caused by reduced activity of JMJC domain-containing lysine demethylases, demonstrating through multiple complementary approaches-including the use of a demethylase inhibitor, overexpression of target-specific demethylases, and analysis from the perspective of cofactors required for JMJC domain-containing demethylases-that decreased lysine demethylase activity constitutes the molecular mechanism underlying the increased H3 methylation levels induced by Chlamydia infection.
(4) By performing ChIP-seq analyses of H3K4me3 and H3K9me3, the study clearly delineates, on a genome-wide scale, how infection leads to increased levels of these epigenomic marks.
Weakness:
(1) Reduction of cofactors such as Fe2+ or a-KG decreases the activity of JMJC-domain-containing lysine demethylases (thereby directly affecting histone H3 lysine methylation). However, these cofactors are also involved in the activities of other epigenetic regulators, such as TET enzymes that contribute to DNA demethylation and SIRT family proteins that mediate histone deacetylation. Therefore, it cannot be excluded that modulation of these factors indirectly leads to the changes in H3 lysine methylation dynamics targeted in this study.
(2) Related to point 1, although overexpression of JMJC-type demethylases has been shown to reduce the Chlamydia infection-induced increase in H3 lysine methylation, it is well known that over production of these enzymes, while target-specific, also leads to a genome-wide reduction of lysine methylation. Thus, a decrease in lysine methylation upon expression of these demethylases does not necessarily demonstrate that the infection-induced increase in H3 lysine methylation is caused by impaired JMJC-type demethylase activity.
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Reviewer #3 (Public Review):
In this manuscript, the authors explore a molecular basis for hypermethylation of histones in epithelial cells infected with the obligate intracellular bacterial pathogen Chlamydia trachomatis. This is of particular interest given that Chlamydia is known to drastically alter host cell gene transcription, and histone hypermethylation would suggest a new way by which Chlamydia interferes with gene expression of its host. Histone methylation was previously implicated in the introduction of dsDNA breaks in infected cells, and the chlamydial effector NUE was reported to methylate histones, but the role of this modification in dictating host cell gene transcription has been unexplored. The authors use a suite of tools to approach this question, including various -omics techniques, genetic approaches, and …
Reviewer #3 (Public Review):
In this manuscript, the authors explore a molecular basis for hypermethylation of histones in epithelial cells infected with the obligate intracellular bacterial pathogen Chlamydia trachomatis. This is of particular interest given that Chlamydia is known to drastically alter host cell gene transcription, and histone hypermethylation would suggest a new way by which Chlamydia interferes with gene expression of its host. Histone methylation was previously implicated in the introduction of dsDNA breaks in infected cells, and the chlamydial effector NUE was reported to methylate histones, but the role of this modification in dictating host cell gene transcription has been unexplored. The authors use a suite of tools to approach this question, including various -omics techniques, genetic approaches, and biochemical assays. Overall, the manuscript provides many interesting pieces of data, though some of them are difficult to reconcile, which may reflect methodological hurdles that are not fully addressed in the current version of the manuscript. My major concerns regard the rationale/interpretation for various mechanistic experiments and that the heterogeneity of the histone hypermethylation phenotype is not addressed which I believe may explain some apparent inconsistencies in the results.
Using an immunofluorescent approach, the authors show that a subpopulation of the nuclei in Chlamydia-infected cells (~10-20%) exhibit high amounts of methylated histone species. This occurs during the late stages of infection, near the time when Chlamydia would lyse the host cell and positively correlates with bacterial burden. Accordingly, halting chlamydial growth blocks the onset of histone hypermethylation. Exogenously supplying cofactors for histone demethylases, the low activity of which is implicated in the histone hypermethylation phenotype, reduces histone hypermethylation. In general, these data are compelling and raise interesting questions about the role of histone methylation in governing chlamydial egress from infected cells. Interestingly, these behaviors seem to arise independently of NUE, the secreted chlamydial histone methyltransferase, supporting the notion that a metabolic reprogramming may underlie the hypermethylation phenomenon.
As noted above, the authors propose that hypermethylation arises due to decreased demethylase activity in infected cells. However, the data do not conclusively support this interpretation. For example, the approaches used to probe demethylase activity rely on (i) a direct biochemical measure of demethylase activity, (ii), pharmacological inhibition of demethylase, and (iii) heterologous expression of a specific demethylase. With the exception of (i), these approaches would be expected to alter histone methylation regardless of the source. That is, inhibition of demethylases should increase histone methylation regardless of whether the source of methylation is increased methylase or decreased demethylase activity. Similarly, overexpression of a demethylase would be expected to reduce cognate histone methylation arising either from increased methylase or decreased demethylase activity.
Moreover, the authors report that the effect of the demethylase inhibitor on histone hypermethylation is significantly potentiated by infection, suggesting that infected cells have greater methylase activity than uninfected cells, because the latter barely respond to the presence of demethylase inhibitor. In other words, a dramatic increase in histone methylation in the presence of demethylase inhibitor is most parsimoniously explained by increased methylation (no longer being removed by demethylase), not decreased demethylation (which would be analogous to treatment with demethylase inhibitor). The authors do not directly assay methylase activity. These concerns extend to the rationale used to justify experiments with infected mice, which the authors treat with the demethylase inhibitor.
The authors perform experiments to characterize the consequence of hypermethylation genome-wide. Because the authors do not enrich for those cells which exhibit histone hypermethylation, the results reflect the mixed population, and therefore presumably dilute out important signal related to the phenomena under investigation. For example, the proteomic analysis of post-translational modifications identifies only one methylated histone species, whereas the immunofluorescent approach shows consistent effects across five different methylated histone species. Moreover, the chromatin immunoprecipitation analysis indicates that there is unexpectedly a lower density of methylated histones at regions which are also enriched in uninfected cells. The authors argue that this suggests increased methylation is happening "outside" of these histone-dense regions, but direct evidence in support of this claim is lacking.
In sum, this paper provides compelling evidence in support of the notion that histones are hypermethylated at various residues late in chlamydial infection, that this process is modulated by known cofactors of demethylases, and is the result of high levels of bacterial replication in the cell. That histone hypermethylation governs host gene transcription during chlamydial infection suggests a relatively novel mechanism by which Chlamydia subverts the host cell to establish a replicative niche or egress to infect a new cell. The information obtained regarding the methylation status of host proteins and host gene transcription controlled by a metabolic cofactor during infection will be a useful resource for other researchers. However, in the current version of the manuscript, the mechanistic basis for these behaviors is relatively unclear.
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Author response:
Reviewer #1 (Public Review):
This study by Charendoff et al provides interesting observations related to global histone hypermethylation in host cells, during Chlamydia trachomatis infections. The core observation they report is that the host histones are highly hypermethylated during infection, and this appears to be an amplifying effect due to continuous inhibition of demethylases, in part due to a metabolic shift in the host where succinate amounts (which inhibit demethylases) increases. The authors claim specifically due to the bacteria, since antibiotic treatment prevents histone hypermethylation (but leaves you wondering about cause/consequence correlations).
The core observation of hyper methylation is very interesting, and well documented. There are a number of points to consider though in order to fully …
Author response:
Reviewer #1 (Public Review):
This study by Charendoff et al provides interesting observations related to global histone hypermethylation in host cells, during Chlamydia trachomatis infections. The core observation they report is that the host histones are highly hypermethylated during infection, and this appears to be an amplifying effect due to continuous inhibition of demethylases, in part due to a metabolic shift in the host where succinate amounts (which inhibit demethylases) increases. The authors claim specifically due to the bacteria, since antibiotic treatment prevents histone hypermethylation (but leaves you wondering about cause/consequence correlations).
The core observation of hyper methylation is very interesting, and well documented. There are a number of points to consider though in order to fully substantiate the findings, and close out loose ends. My comments are broad - and built around the interpretations (vs the data presented).
(1) Related to observations coming Fig 1C etc, and connecting to Fig 3 - the hyper methylation appears to be across different protein arg/lys residues - and is not histone specific. So, is it just a consequence of high SAM pools and flux in infected cells? i.e. the bacterial infection increases SAM pools in cells, and provides an increase in substrate pools for the methyltransferases, leading to protein hyper methylation. The approach used here only measures steady-state SAM amounts (and not SAM flux or utilisation).
For example, reduced SAM amounts in nuclei could be due to increased utilisation of SAM. The experiments done with the demethylase does not actually answer this question - if you decrease demethylase activity, you will get an increase in net methylation. The authors see an increase in net methylation in the infected cells - this would suggest that in addition (or perhaps primarily) to reduced demethylase activity, there could be much higher SAM utilisation/flux. Again, the over expression of JMJ proteins does not resolve this problem.
This is an important point. Indeed, one limitation of the initial version of the paper was that we had measured SAM concentration only at one time point (40 hpi) and on the whole population. During revision we used a ratiometric sensor to measure SAM concentration in cells (PMID 34937909). We observed cell-to-cell heterogeneity in SAM levels in HeLa cells, as previously reported in other cell lines. Chlamydia inclusions develop asynchronously, which allows to observe, 40 hpi, a continuum of early (low bacterial load) to late (high bacterial load) stages of infection. We observed no correlation between bacterial load and SAM level, and SAM levels were globally similar when comparing infected and non-infected cells. This experiment strongly supports the hypothesis that protein hypermethylation is not due to an increase in SAM during infection. The data were added in the New Fig. 3. Note that the former Fig. 3 is now split into New Fig. 3 and New Fig. 4.
(2) Adding to this - what happens to SAM pools in the cells treated with the inhibitors? This actually may not look like the slightly reduced SAM pool observed in infected cell nuclei. Also, what is the SAM/SAH ratio (a very useful indicator of methylation activity).
Based on the high cell-to-cell heterogeneity of SAM levels observed with the ratiometric probe, we reasoned that measuring SAM/SAH ratio without single cell resolution would not bring crucial information. Also, the discrepancy between data displayed in new Fig. 3A (nuclear extracts) and 3C (live cell imaging) indicate that SAM might be less stable in cellular extracts from infected cells compared to non-infected ones, which would complicate the interpretation of the data. Therefore, we did not implement LC-MS/MS on nuclear extracts to measure SAM/SAH ratio.
(3) There is a correlation/implication issue here in Fig 2 - cells with C. trachoma's infection show hyper methylation. But these are the only cells with high C. trachomatis. So it is a bit ingenious to say that histone hyper methylation correlates with bacterial proliferation. The cells without bacteria don't have hyper methylation - and that does not have anything to do with the bacterial proliferation.
In Fig. 2B, we compared the methylation signal within the population of infected cells only (excluding the uninfected cells). We edited the text to clarify this point. “We observed that, within the population of infected cells, the sum intensity of the mCherry signal was higher in cells that displayed hypermethylation of H3K9me3 than in cells with low level of H3K9me3, indicating that histone hypermethylation correlated with bacterial load (Fig. 2B).”
(4) The claim that demethylase activity is down in infected cells again comes primarily from the increased succinate (2-fold) amounts in infected nuclei - and then correlated with experiments where succinate, (permeable) a-KG are supplemented in excess. While I personally like the hypothesis that the hypermethylation might be a result of an imbalance in cofactors (succinate vs a-KG) in infected cells, the data presented is very premature to make that conclusion. Again, steady state measurements of only succinate cannot provide a clear answer to that question. For example, is there a clear allocation/flux difference (between a-KG, and leading out to glutamate/glutamine, vs flux through the TCA and increased succinate accumulation? Is there a bottleneck/build-up of succinate in cells that might lead to the increase in nuclei? This also opens another direction of possible regulation - increased histone succinylation. When you see a large increase in succinate in the nucleus, before looking at demethylase activity - it becomes obvious if succinate itself increases histone succinylation (through HATs).
Our work confirms the accumulation of succinate in cells infected by C. trachomatis, previously reported in Rother et al 2018. The reason for this accumulation remains to be investigated in detail. We have previously shown that OxPhos is relatively stable in infected cells (PMID 35931114), indicating that the flux through the TCA of the eukaryotic host proceeds normally. As mentioned in our discussion, the TCA of the bacteria is disrupted with several enzymes missing, although not in the step immediately downstream of succinate/fumarate production. Still, synthesis of succinate and fumarate (fumarate accumulation was observed in the Rother 2018 study) by bacterial enzymes might contribute to their accumulation in infected cells. The approach we chose to measure methylation at the proteome level is not suitable to look for histone succinylation, because of the diversity of post translational modifications on histones, which occur in combinations. However, following on this reviewer’s comment, we reanalysed the proteomic data to compare protein succinylation levels in infected and non-infected samples. We detected 41 succinylated peptides in the infected samples, against 23 in the uninfected samples. For many of these, we did not have quantitative data in all condition and only one protein, transportin 1 (TNPO1), reached statistical significance, with a 4-fold increase in succinylation in infected samples. Thus, while essentially qualitative, this analysis fully supports the hypothesis that succinate accumulates in infected cells. These data were added to Table S1 and to the result section.
(5) What might the authors hypothesise about why this hyper methylation happens? It appears in some ways that hyper methylation happens - potentially due to a metabolic bottleneck that the bacteria triggers (and there is a build-up of SAM and/or succinate, and altered flux out of a-kg). The methylation is just a visible outcome - but may not be central to pathogenesis or viability.
We discussed this question in the penultimate paragraph of the discussion by giving some elements of answer to the question: “Does it benefit the host or the bacteria? ». In our study, we showed that protein hypermethylation affected the transcriptional response of the host. We did not investigate whether the activity of some of the host proteins engaged in the response to infection were affected. It might be the case, considering that methylation is a common PTM regulating protein’s activity. Still, we agree with this reviewer that hypermethylation might not be central to pathogenesis or viability. Addressing this question would require a complex model in which protein methylation levels could be controlled experimentally.
Reviewer #2 (Public Review):
Strengths:
(1) Because the study compares genuinely infected cells with uninfected cells within the same infected cell population, it enables a clearer and more rigorous comparison.
(2) By using multiple Chlamydia species and cells from multiple host species (human and mouse), and obtaining consistent findings across these systems, the study demonstrates the generality of bacterium-induced epigenomic alterations.
(3) The study shows that the epigenomic changes are caused by reduced activity of JMJC domain-containing lysine demethylases, demonstrating through multiple complementary approaches-including the use of a demethylase inhibitor, overexpression of target-specific demethylases, and analysis from the perspective of cofactors required for JMJC domain-containing demethylases-that decreased lysine demethylase activity constitutes the molecular mechanism underlying the increased H3 methylation levels induced by Chlamydia infection.
(4) By performing ChIP-seq analyses of H3K4me3 and H3K9me3, the study clearly delineates, on a genome-wide scale, how infection leads to increased levels of these epigenomic marks.
Weakness:
(1) Reduction of cofactors such as Fe2+ or a-KG decreases the activity of JMJC-domaincontaining lysine demethylases (thereby directly affecting histone H3 lysine methylation). However, these cofactors are also involved in the activities of other epigenetic regulators, such as TET enzymes that contribute to DNA demethylation and SIRT family proteins that mediate histone deacetylation. Therefore, it cannot be excluded that modulation of these factors indirectly leads to the changes in H3 lysine methylation dynamics targeted in this study.
Indeed, reduction of the concentration of Fe2+ and aKG is expected to have other consequences in addition to the inhibition of JMJC-domain containing lysine demethylases on which we focus in this study. As a matter of fact, we reported a decrease in the methylation level of host DNA in infected cells, and we brought some elements that might explain the discrepancy between DNA and histone methylation status in the discussion (e.g., infected cells display enhanced expression of GADD45, which recruit TET enzymes and thus facilitate DNA demethylation). This example illustrates the complexity of host/pathogen interplay, which affect many parameters simultaneously. Indeed, we cannot rule out that modulation of enzymatic activities other than JMJC-domain containing lysine demethylase contribute significantly to the hypermethylation phenotype.
(2) Related to point 1, although overexpression of JMJC-type demethylases has been shown to reduce the Chlamydia infection-induced increase in H3 lysine methylation, it is well known that over production of these enzymes, while target-specific, also leads to a genome-wide reduction of lysine methylation. Thus, a decrease in lysine methylation upon expression of these demethylases does not necessarily demonstrate that the infection-induced increase in H3 lysine methylation is caused by impaired JMJC-type demethylase activity.
We fully agree. We included this experiment to show that increasing the expression of one demethylase only restored demethylation of its cognate target. This support the hypothesis that if the hypermethylation is due to poor demethylase activity, it is likely that several demethylases show impaired activity (as opposed to a scenario in which failure of activity of a single demethylase would indirectly affect all other methylation marks).
Reviewer #3 (Public Review):
In this manuscript, the authors explore a molecular basis for hypermethylation of histones in epithelial cells infected with the obligate intracellular bacterial pathogen Chlamydia trachomatis. This is of particular interest given that Chlamydia is known to drastically alter host cell gene transcription, and histone hypermethylation would suggest a new way by which Chlamydia interferes with gene expression of its host. Histone methylation was previously implicated in the introduction of dsDNA breaks in infected cells, and the chlamydial effector NUE was reported to methylate histones, but the role of this modification in dictating host cell gene transcription has been unexplored. The authors use a suite of tools to approach this question, including various -omics techniques, genetic approaches, and biochemical assays. Overall, the manuscript provides many interesting pieces of data, though some of them are difficult to reconcile, which may reflect methodological hurdles that are not fully addressed in the current version of the manuscript. My major concerns regard the rationale/interpretation for various mechanistic experiments and that the heterogeneity of the histone hypermethylation phenotype is not addressed which I believe may explain some apparent inconsistencies in the results.
We thank this reviewer for insightful comments. We address these two major concerns during revision and bring some elements in our responses below.
Using an immunofluorescent approach, the authors show that a subpopulation of the nuclei in Chlamydia-infected cells (~10-20%) exhibit high amounts of methylated histone species. This occurs during the late stages of infection, near the time when Chlamydia would lyse the host cell and positively correlates with bacterial burden.
Accordingly, halting chlamydial growth blocks the onset of histone hypermethylation. Exogenously supplying cofactors for histone demethylases, the low activity of which is implicated in the histone hypermethylation phenotype, reduces histone hypermethylation. In general, these data are compelling and raise interesting questions about the role of histone methylation in governing chlamydial egress from infected cells. Interestingly, these behaviors seem to arise independently of NUE, the secreted chlamydial histone methyltransferase, supporting the notion that a metabolic reprogramming may underlie the hypermethylation phenomenon.
As noted above, the authors propose that hypermethylation arises due to decreased demethylase activity in infected cells. However, the data do not conclusively support this interpretation. For example, the approaches used to probe demethylase activity rely on (i) a direct biochemical measure of demethylase activity, (ii), pharmacological inhibition of demethylase, and (iii) heterologous expression of a specific demethylase. With the exception of (i), these approaches would be expected to alter histone methylation regardless of the source. That is, inhibition of demethylases should increase histone methylation regardless of whether the source of methylation is increased methylase or decreased demethylase activity. Similarly, overexpression of a demethylase would be expected to reduce cognate histone methylation arising either from increased methylase or decreased demethylase activity.
We agree with the reviewer’s comments. The experiment using pharmacological inhibitors (ii) show that infected cells are sensitized to these inhibitors but doesn’t provide direct mechanistic insight. The experiment using heterologous expression of demethylases (iii) was included to show that increasing the expression of one demethylase only restored demethylation of its cognate target. This supports the hypothesis that several demethylases show impaired activity (as opposed to a scenario in which failure of activity of a single demethylase would indirectly affect all other methylation marks).
The most direct evidence for impaired demethylase activity come from the direct measure of demethylation of H3K4me3 in nuclear extract (i). It is strengthened by indirect evidence that metabolite concentrations hinder demethylase activities late in infection: 1/ iron and DMKG supply diminish hypermethylation of histone lysine residues 2/ succinate levels (a competitor of aKG) are two-fold higher in nuclei isolated from infected cells. This latter finding was confirmed during revision as we identified more succinylated proteins in infected samples compared to non-infected ones.
We also considered the possibility that infected cells displayed increased histone methyl transferase (HMT) activity. This would be compatible with decrease KDM activity and could contribute to the histone hypermethylation. Unfortunately, this hypothesis cannot be tested directly (as we did for the measure of H3K4me3 demethylation activity). Indeed, SAM is notoriously labile and in vitro assays to measure HMT require to add exogenous SAM to cell extracts to detect any HMT activity, which would not allow us to test activity based on endogenous SAM levels.
Instead, we used a ratiometric sensor to measure SAM concentration in cells (PMID 34937909). Chlamydia inclusions develop asynchronously, which allows to observe, 40 hpi, a continuum of early (low bacterial load) to late (high bacterial load) stages of infection. There was no correlation between bacterial load and SAM level, and this level was globally similar when comparing infected and non-infected cells. This experiment supports our hypothesis that protein hypermethylation is not due to an increase in SAM during infection.
This experiment was also very interesting because it revealed a high cell-to-cell heterogeneity in SAM levels in HeLa cells. Thus, in some cells, SAM might be limiting, which could explain why only a fraction of cells display histone hypermethylation.
Still, we cannot fully rule out the possibility that increase in SAM availability late in the infectious cycle in some cells, and is immediately consumed through protein methylation, resulting in no net [SAM] increase. The discussion was expanded to take these comments into consideration.
Altogether, we think that the evidence of decrease KDM activities in infected cells late in infection are strong. Our data do not rule out the possibility that additional mechanisms may contribute.
Moreover, the authors report that the effect of the demethylase inhibitor on histone hypermethylation is significantly potentiated by infection, suggesting that infected cells have greater methylase activity than uninfected cells, because the latter barely respond to the presence of demethylase inhibitor. In other words, a dramatic increase in histone methylation in the presence of demethylase inhibitor is most parsimoniously explained by increased methylation (no longer being removed by demethylase), not decreased demethylation (which would be analogous to treatment with demethylase inhibitor). The authors do not directly assay methylase activity. These concerns extend to the rationale used to justify experiments with infected mice, which the authors treat with the demethylase inhibitor.
The observation that the same concentration of JIB-04 leads to an increase of histone methylation in infected cells and not in non-infected cells, is coherent with the data showing that aKG or iron supply diminish histone hypermethylation in infected cells. Indeed, the inhibitor is taken up similarly by infected and uninfected cells but the potency of the inhibitor will depend partly on levels of iron, aKG and succinate found in the cellular milieu so same concentration of inhibitor may inhibit demethylase activity in cells with higher succinate and/or low aKG and low iron but fail to inhibit demethylase activity in cells with higher iron or aKG or lower succinate. In other words, high iron, high aKG or low succinate will “buffer” JIB-04 and make it less potent since JIB-04 partly acts by competing with the iron (competitively) and the aKG (mixed competitive inhibition) PMID 23792809. The same phenomenon is expected for SD70 and TACH101 that share aspects of the mode of action of JIB-04 regarding partly competing for aKG and/or iron in the catalytic site.
The authors perform experiments to characterize the consequence of hypermethylation genome-wide. Because the authors do not enrich for those cells which exhibit histone hypermethylation, the results reflect the mixed population, and therefore presumably dilute out important signal related to the phenomena under investigation. For example, the proteomic analysis of post-translational modifications identifies only one methylated histone species, whereas the immunofluorescent approach shows consistent effects across five different methylated histone species. Moreover, the chromatin immunoprecipitation analysis indicates that there is unexpectedly a lower density of methylated histones at regions which are also enriched in uninfected cells. The authors argue that this suggests increased methylation is happening "outside" of these histone-dense regions, but direct evidence in support of this claim is lacking.
The caveat of bulk analyses as opposed to single cell resolution is indeed important to consider when analysing the chIP-seq data and we emphasized this point in the revised manuscript. We could have sorted the cells with high bacterial burden; this would probably have given stronger differences between the two samples. Still, the change in distribution of H3K4me3 in infected samples was very clear and statistically significant. A change in H3K9me3 distribution would be more difficult to catch, as the mark is more widespread.
In sum, this paper provides compelling evidence in support of the notion that histones are hypermethylated at various residues late in chlamydial infection, that this process is modulated by known cofactors of demethylases, and is the result of high levels of bacterial replication in the cell. That histone hypermethylation governs host gene transcription during chlamydial infection suggests a relatively novel mechanism by which Chlamydia subverts the host cell to establish a replicative niche or egress to infect a new cell. The information obtained regarding the methylation status of host proteins and host gene transcription controlled by a metabolic cofactor during infection will be a useful resource for other researchers. However, in the current version of the manuscript, the mechanistic basis for these behaviors is relatively unclear.
We thank this reviewer for constructive feedback. We believe that the mechanistic conclusions of our report have been strengthened during revision with additional experiments and text clarification.
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