TPR is required for cytoplasmic chromatin fragment formation during senescence

  1. Bethany M Bartlett
  2. Yatendra Kumar
  3. Shelagh Boyle
  4. Tamoghna Chowdhury
  5. Andrea Quintanilla
  6. Charlene Boumendil
  7. Juan Carlos Acosta
  8. Wendy A Bickmore

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Abstract

During oncogene-induced senescence there are striking changes in the organisation of heterochromatin in the nucleus. This is accompanied by activation of a pro-inflammatory gene expression programme – the senescence-associated secretory phenotype (SASP) – driven by transcription factors such as NF-κB. The relationship between heterochromatin re-organisation and the SASP has been unclear. Here, we show that TPR, a protein of the nuclear pore complex basket required for heterochromatin re-organisation during senescence, is also required for the very early activation of NF-κB signalling during the stress-response phase of oncogene-induced senescence. This is prior to activation of the SASP and occurs without affecting NF-κB nuclear import. We show that TPR is required for the activation of innate immune signalling at these early stages of senescence and we link this to the formation of heterochromatin-enriched cytoplasmic chromatin fragments thought to bleb off from the nuclear periphery. We show that HMGA1 is also required for cytoplasmic chromatin fragment formation. Together these data suggest that re-organisation of heterochromatin is involved in altered structural integrity of the nuclear periphery during senescence, and that this can lead to activation of cytoplasmic nucleic acid sensing, NF-κB signalling, and activation of the SASP.

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  1. Version published to 10.7554/elife.101702 on eLife
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    Reply to the reviewers

    Reviewer #1

    i) "Enhancers dependent on TPR during senescence are enriched for binding sites of inflammatory transcription factors". *Proximity to genes does not confirm an enhancer role for that gene, although Tasdemir et al., 2016 suggested this. At that time, HI-C and Hi-CHiP techniques were not well-established. Nowadays, without combining HI-C and H3K27ac ChIP, Hi-ChIP alone cannot definitively identify actual enhancer regions. If we repeatedly use the Tasdemir et al., 2016 map, we risk incorrect mapping of enhancers of SASP. The authors should either use other public Hi-C databases to map the enhancer of SASP or temper their conclusions about enhancers. Otherwise, this could set a precedent for the SASP enhancer region that might not be entirely accurate. *

    The enhancer mapping for SASP is outdated, as advancements in Hi-C have significantly developed this area. Therefore, the claimed enhancers of SASP may not be accurate.

    __Response: __We agree with the reviewer that enhancers are not easy to define, or to pair with their target gene(s). Indeed, we would argue that even combined HI-C and H3K27ac does not define enhancers or enhancer-gene pairs and that the gold-standard evidence for an enhancer is genetics – does its deletion/mutation abrogate gene activation. We would also point out that we did not actually use the Tasdemir data to call enhancers. In response to the reviewer’s comment, we will temper our terminology and now refer to our inter-and intra-genic ATAC-seq peaks only as “putative enhancers”.

    ii) “Many of these include putative enhancers located close to key SASP genes, such as IL1B and IL8 (Figure 1D).” I have the same concern as mentioned above (i). However, I am interested in knowing the other key SASP genes where DNA is accessible near the genes. A supplementary table listing key SASP genes along with their distances to the TSS and affected by TPR knock-down would be helpful.

    __Response: __We thank the reviewer for this suggestion. We will provide tables listing the TPR dependent, senescent specific ATAC-seq peaks that are close to genes associated with the ‘positive regulation of inflammatory response’, ‘cytokine activity’ and ‘cytokine receptor binding’ gene ontology terms which were significant in the GREAT analysis, and which includes many SASP genes. We will also provide distances of these regions from the associated genes.

    iii) "As we previously reported, knockdown of TPR (siTPR) in RAS cells blocks SAHF formation, but it also results in reduced nuclear localisation (decreased nucleocytoplasmic ratio) of NF-κB, consistent with decreased NF-κB activation (Figure 2A and B, Figure S2A)." TPR is required for CCF, SASP, and SAHF. The relationship between CCF and SASP is well established, but the relationship between SAHF and CCF/SASP remains elusive. Both SAHF and CCF are enriched with heterochromatin markers, suggesting that CCF might originate from SAHF. However, this has not been confirmed. Do the authors think that SAHF is a prerequisite for CCF in the OIS model, or is it an independent event?

    Response: We agree with the reviewer that CCFs likely originate from SAHF. Whilst we cannot definitively prove thisin our ER-Ras OIS model, in the revised manuscript we intend to further investigate the relationship between SAHF and CCF by knocking down HMGA1 during RAS-induced senescence. Like TPR, HMGA1 depletion is known to lead to loss of SAHF (Narita et al., Cell, 2006) but, unlike TPR, HMGA1 is a chromatin protein enriched on heterochromatin itself. We will assess whether loss of HMGA1 also abrogates CCF formation.

    iv) The authors suggested that "it is plausible that the decrease in CCFs produced during the early phases of OIS upon TPR knockdown may be caused by an increase in the stability of the nuclear periphery due to the heterochromatin that remains there when SAHF are not formed." I do not completely agree with this explanation because CCF starts forming at day 3-4 but culminates at later time points. According to Figure 5A, only 5-6% of cells are positive for CCFs on day 5. What happens on day 8? By day 8, the percentage of CCF-positive cells could be 20-25%, or the number of CCFs per cell might be 0.2-0.3. If TPR is not required for CCF formation at this stage, then linking CCF to SASP at day 8 becomes critical. This suggests that another mechanism might be driving SASP expression and that TPR could be regulating downstream signaling of CCF. It is possible that changes in nuclear pore density affect the localization of cGAS from the nucleus to the cytoplasm.

    Response: In our hands and using this IMR90 ER-RAS system, CCF formation decreases later in senescence (d8 - only 2% of cells) hence our focus on early timepoints after oncogenic RAS activation. At later timepoints, cGAS activation is also mediated by retrotransposons (de Cecco et al., Nature, 2019; Liu et al., Cell, 2023), as well as leakage of mitochondrial DNA (Victorelli et al., Nature, 2023; Chen et al., Nat. Comms, 2024), and so it is difficult to disentangle the net contribution of these three inputs.

    v) Additionally, the authors did not address what happens in the later stages of CCF formation in the absence of TPR. If TPR is not required for CCF formation at later stages, it fails to explain the downstream processes at these time points adequately. This suggests that TPR may also have another mechanism of SASP regulation independent of CCF formation.

    __Response: __In our cellular system CCFs precede the SASP - CCFs are already present at day 3 but SASP factors are not secreted until day 5. However, CCFs are not necessarily required for maintenance of the SASP. Once initiated the SASP is maintained by cytokine feedback loops.

    …………

    Reviewer #2:

    1. The claim that TPR knockdown does not affect NFkappaB nuclear translocation indeed stands, but it would be nice if the authors also compared data across conditions in Fig. 2F, i.e. siCTRL+Ras CM versus siTPR+Ras CM in RAS cells and provided a p-value as it seems to me that there is some dampening of translocation intensity, which is clearly not the case for STOP cells. The authors focus on this for d3 and d5, but it seems to be also the case for later time points.

    __Response: __As basal NF-κB translocation is lower in RAS cells on TPR knockdown, we would expect a dampening in NF-κB translocation between siCTRL+RAS CM and siTPR+Ras CM regardless of whether there is a transportation defect. Consistent with this, the p-value for this comparison is significant, but we did not show it because it is not important in considering whether NF-κB nuclear translocation is impeded by TPR knockdown, which is the focus here. We will add a table with median nuclear:cytoplasmic NF-κB ratios and 95% confidence intervals to make the changes in basal level (treatment with STOP CM) clearer.

    Also, a comment based on literature or from the authors previous work on TPR, on the extent to which the structural integrity of the nuclear basket is at all affected upon TPR depletion would be helpful for data interpretation.

    __Response: __In the revised manuscript we will refer to the literature showing that TPR is the final component added to the nuclear pore and that its absence does not affect localisation of NUP153 to the nuclear basket (Hase and Cordes., Mol. Biol. Cell 2003; Aksenova et al., Nat Comms, 2020).

    Magnification of representative cells per each condition in Fig. 2E would be welcome.

    __Response: __We will provide a revised figure 2E with the magnifications as requested.

    Regarding the data in Figs 3 and S3: I am a bit confused about how the obviously decreased NFkappaB nuclear signal (e.g., in Fig. 3D) does not translate into a skewed N/C ratio (e.g., in Fig. 3C)? The western blots indicate that overall NFkappaB levels remain essentially unchanged? Am I missing something?

    __Response: As stated in the Methods section, we used a 50-pixel expansion of the detected nuclear area as our cytoplasmic area in the analysis (see image below). This was because we found detecting and segmenting the whole cytoplasmic area in the NF-κB channel to be unreliable. At day 3 and 5, the decrease in NF-κB nuclear signal in RAS cells on TPR knockdown was accompanied by a decrease in signal in the portion of the cytoplasm closest to the nucleus. This led to no change in the nuclear:cytoplasmic ratio. We believe the redistribution of NF-κB closer to the nucleus in the RAS siCTRL sample indicates early activation and will make this clearer in the revised text. We will also quantify the NF-κB western blots (see point 5), to help clarification of this issue.____ __

    Also, along these lines, d8 western blots seem to portray an overall drop in NFkappaB levels. Is this indeed so? Can the authors maybe quantify their blots' replicates and provide a box plot and statistical testing?

    Response: We will provide quantification for the NF-κB western blots, though box plots would not be appropriate as we only have two replicates.

    Regarding the ATAC-seq data from d3, I think it could be mined a bit more. For example, compare to d8 (which the authors have apparently done, but don't present in detail) and discuss which are these early regions that also become accessible by d3 and what kind of genes and motifs are associated with them. Moreover, the focus in Fig. S3E is on ATAC sites shared with d8; how about d3-specific ones? How many of these are there (if any) and how might they be affected?

    __Response: __As shown in Table S2, TPR knockdown did not cause any changes in chromatin accessibility at day 3, so there are no day 3 specific TPR dependent peaks. We will edit the text to make this clearer. We will carry out motif analysis and GREAT analysis on the day 3 peaks that become accessible in RAS cells but are not accessible in STOP (RAS-specific peaks).

    I trust that the authors quantified their STING blots for the conclusions they present, but since it is difficult to assess these confidently by eye, again, some quantification plots would be welcome in Figs 4C,D and S4D,E.

    __Response: __We will provide quantification for the STING western blots.

    As controls for Fig. 5, it would be interesting to see if active histone readouts also mark CCFs in this system.

    __Response: __Ivanov et al., J. Cell Biol., 2013 showed the absence of H3K9 acetylation from chromatin in CCFs. Further exploration of the types of chromatin/sequences in CCFs is outside the scope of our current manuscript.

    *The POM121 channel in Fig. 5C appears to have some small signal foci in the cytoplasm; could these be small CCFs? More generally, the authors focus on these large blobs that only appear in

    __Response: __The small signal foci the reviewer is highlighting are background from the POM121 antibody staining rather than CCFs – they do not show DAPI staining, and similar foci are evident in non-senescent cells where CCFs are generally not present. Our unpublished data (see response to Reviewer 1, point iv) from day 8 cells shows that only ~2% of senescent cells are positive for CCF regardless of TPR knockdown, which is a similar number to that observed in non-senescent cells at earlier timepoints. Thus, in our hands CCF formation occurs earlier, triggering the SASP, rather than at day 8 when the SASP is already established and reinforced through positive feedback cytokine signalling.

    I wonder if there is a simple experiment the authors could do to test if this mechanism is only linked to senescence, specifically oncogene-induced senescence? I don't think this is needed to support the conclusions drawn here, but it could significantly broaden the scope of their discovery of, for example, this was true in other senescence models or during proinflammatory activation in general?

    __Response: __These are interesting suggestions, but setting up, characterising and quantifying other senescence models will take a substantial amount of time that would be outside the scope of our current manuscript.

    ………….

    Reviewer #3

    1. The study uses a single cell strain IMR90 undergoing a single form of senescence, induced by activated Ras. To show the generalizability of the finding, the authors are advised to inhibit TPR in other forms of senescence in addition to IMR90. For example, IR or etoposide induces greater amount of CCF than in OIS of IMR90. BJ, MEFs, and ARPE-19 senescence also show prominent CCF.

    __Response: __These are interesting suggestions, but as we responded to reviewer 2, setting up, characterising and quantifying other senescence models will take a substantial amount of time that would be outside the scope of our current manuscript.

    To convincing show the CCF pathway is involved, the authors need to measure the activity of cGAS-STING pathway. Including cGAMP ELISA will be informative.

    __Response: __We thank the reviewer for this suggestion, and we will try to include this assay in our revised manuscript.

    The authors used conditioned media to show that TPR KD does not directly affect NFkB nuclear translocation. While this is helpful, conditions other than senescence will be more direct. For example, TNFa treatment or poly I:C transfection induces efficient NFkB nuclear translocation in IMR90 cells.

    __Response: __This experiment (Fig. 2EF) was designed to simply show that knocking down TPR does not impair the ability of activated NFkB to enter the nucleus, it is not about senescence per se. Indeed, this is why we included the addition of SASP (RAS) conditioned media to non-senescence STOP cells in Fig. 2. We do not think investigating other methods of activating NFkB would add more to the question of whether TPR loss abrogates NFkB nuclear import.

    Fig. 4C and Fig. S4D are identical.

    Response: Though these STING immunoblots look similar; in fact they are not identical. Below we attach the raw original image in which both biological replicates (Fig 4C and S4D) for Day 3 were run on the same gel as proof of this claim.

    Figure legend for Fig. S4F is mislabeled.

    __Response: __We will correct this.

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    Referee #3

    Evidence, reproducibility and clarity

    Bartlett et al reported that knockdown of TPR inhibits CCF and NFkB during oncogene-induced senescence in IMR90 cells. The manuscript is well-written, and the results are clear and convincing. I have the following suggestions.

    1. The study uses a single cell strain IMR90 undergoing a single form of senescence, induced by activated Ras. To show the generalizability of the finding, the authors are advised to inhibit TPR in other forms of senescence in addition to IMR90. For example, IR or etoposide induces greater amount of CCF than in OIS of IMR90. BJ, MEFs, and ARPE-19 senescence also show prominent CCF.
    2. To convincing show the CCF pathway is involved, the authors need to measure the activity of cGAS-STING pathway. Including cGAMP ELISA will be informative.
    3. The authors used conditioned media to show that TPR KD does not directly affect NFkB nuclear translocation. While this is helpful, conditions other than senescence will be more direct. For example, TNFa treatment or poly I:C transfection induces efficient NFkB nuclear translocation in IMR90 cells.

    Minor:

    1. Fig. 4C and Fig. S4D are identical.
    2. Figure legend for Fig. S4F is mislabeled.

    Significance

    This study builds on the group's prior publication that knockdown of TPR inhibits SAHF and SASP. The current study finds that the underlying mechanism is via CCF-STING-NFkB pathway. Overall, the study broadens our understanding of CCF and SASP in senescence, and will be of general interest to the senescence field.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript by Bartlett et al. revisits the role of the nuclear pore component, TPR, in OIS to uncover its contribution to NFkappaB activation magnitude via the control of chromatin fragment release from the senescent nucleus. The flow of the manuscript is very good and the conclusions are supported by clear experimental evidence. This is an overall mature manuscript, and I offer below some comments that might help the authors streamline their message even more:

    • The claim that TPR knockdown does not affect NFkappaB nuclear translocation indeed stands, but it would be nice if the authors also compared data across conditions in Fig. 2F, i.e. siCTRL+Ras CM versus siTPR+Ras CM in RAS cells and provided a p-value as it seems to me that there is some dampening of translocation intensity, which is clearly not the case for STOP cells. The authors focus on this for d3 and d5, but it seems to be also the case for later time points.
    • Also, a comment based on literature or from the authors previous work on TPR, on the extent to which the structural integrity of the nuclear basket is at all affected upon TPR depletion would be helpful for data interpretation.
    • Magnification of representative cells per each condition in Fig. 2E would be welcome.
    • Regarding the data in Figs 3 and S3: I am a bit confused about how the obviously decreased NFkappaB nuclear signal (e.g., in Fig. 3D) does not translate into a skewed N/C ratio (e.g., in Fig. 3C)? The western blots indicate that overall NFkappaB levels remain essentially unchanged? Am I missing something?
    • Also, along these lines, d8 western blots seem to portray an overall drop in NFkappaB levels. Is this indeed so? Can the authors maybe quantify their blots' replicates and provide a box plot and statistical testing?
    • Regarding the ATAC-seq data from d3, I think it could be mined a bit more. For example, compare to d8 (which the authors have apparently done, but don't present in detail) and discuss which are these early regions that also become accessible by d3 and what kind of genes and motifs are associated with them. Moreover, the focus in Fig. S3E is on ATAC sites shared with d8; how about d3-specific ones? How many of these are there (if any) and how might they be affected?
    • I trust that the authors quantified their STING blots for the conclusions they present, but since it is difficult to assess these confidently by eye, again, some quantification plots would be welcome in Figs 4C,D and S4D,E.
    • As controls for Fig. 5, it would be interesting to see if active histone readouts also mark CCFs in this system.
    • The POM121 channel in Fig. 5C appears to have some small signal foci in the cytoplasm; could these be small CCFs? More generally, the authors focus on these large blobs that only appear in <6% of cells in d3 and d5. Does this increase by d8? What is the effect of TPR knockdown on CCF numbers at that later time point?
    • I wonder if there is a simple experiment the authors could do to test if this mechanism is only linked to senescence, specifically oncogene-induced senescence? I don't think this is needed to support the conclusions drawn here, but it could significantly broaden the scope of their discovery of, for example, this was true in other senescence models or during proinflammatory activation in general?

    I typically disclose my identity to the authors: A. Papantonis

    Significance

    This is a very clearly written and well-controlled study that addresses a gap in knowledge from the previous work on TPR in senescence. It also brings about a perhaps unexpected effect of a nuclear pore component on NFkappaB signaling that might not necessarily be senescence-specific. As such, I think that the study would be of interest to both the senescence community and to researchers studying inflammatory responses, especially those driven by TNFalpha or IL1A/B.

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    Referee #1

    Evidence, reproducibility and clarity

    DNA damage triggers senescence, inducing chromatin reorganization and SASP activation. The authors previously demonstrated that the TPR nucleoprotein at nuclear pores is crucial for both SAHF formation and SASP activation during senescence. Here they also showed that TPR is required for the formation of cytoplasmic chromatin fragments (CCF), which activate cGAS-STING-TBK1-NF-kB signaling to express SASP. While the mechanistic regulation of CCF formation by TPR remains unclear, their study provides compelling evidence of downstream processes involving CCF. This study offers new insights into CCF formation, suggesting a promising direction for further research. I endorse the manuscript; however, there are several concerns that need addressing before acceptance.

    i) "Enhancers dependent on TPR during senescence are enriched for binding sites of inflammatory transcription factors".

    Proximity to genes does not confirm an enhancer role for that gene, although Tasdemir et al., 2016 suggested this. At that time, HI-C and Hi-CHiP techniques were not well-established. Nowadays, without combining HI-C and H3K27ac ChIP, Hi-ChIP alone cannot definitively identify actual enhancer regions. If we repeatedly use the Tasdemir et al., 2016 map, we risk incorrect mapping of enhancers of SASP. The authors should either use other public Hi-C databases to map the enhancer of SASP or temper their conclusions about enhancers. Otherwise, this could set a precedent for the SASP enhancer region that might not be entirely accurate.

    ii) Many of these include putative enhancers located close to key SASP genes, such as IL1B and IL8 (Figure 1D).

    I have the same concern as mentioned earlier about enhancers. However, I am interested in knowing the other key SASP genes where DNA is accessible near the genes. A supplementary table listing key SASP genes along with their distances to the TSS and affected by TPR knock-down would be helpful.

    iii) "As we previously reported, knockdown of TPR (siTPR) in RAS cells blocks SAHF formation, but it also results in reduced nuclear localisation (decreased nucleocytoplasmic ratio) of NF-κB, consistent with decreased NF-κB activation (Figure 2A and B, Figure S2A)." TPR is required for CCF, SASP, and SAHF. The relationship between CCF and SASP is well established, but the relationship between SAHF and CCF/SASP remains elusive. Both SAHF and CCF are enriched with heterochromatin markers, suggesting that CCF might originate from SAHF. However, this has not been confirmed. Do the authors think that SAHF is a prerequisite for CCF in the OIS model, or is it an independent event?

    iv) The authors suggested that "it is plausible that the decrease in CCFs produced during the early phases of OIS upon TPR knockdown may be caused by an increase in the stability of the nuclear periphery due to the heterochromatin that remains there when SAHF are not formed." I do not completely agree with this explanation because CCF starts forming at day 3-4 but culminates at later time points. According to Figure 5A, only 5-6% of cells are positive for CCFs on day 5. What happens on day 8? By day 8, the percentage of CCF-positive cells could be 20-25%, or the number of CCFs per cell might be 0.2-0.3. If TPR is not required for CCF formation at this stage, then linking CCF to SASP at day 8 becomes critical. This suggests that another mechanism might be driving SASP expression and that TPR could be regulating downstream signaling of CCF. It is possible that changes in nuclear pore density affect the localization of cGAS from the nucleus to the cytoplasm.

    Significance

    The authors previously demonstrated that the TPR nucleoprotein at nuclear pores is crucial for both SAHF formation and SASP activation during senescence. Here they also showed that TPR is required for the formation of cytoplasmic chromatin fragments (CCF), which activate cGAS-STING-TBK1-NF-kB signaling to express SASP. While the mechanistic regulation of CCF formation by TPR remains unclear, their study provides compelling evidence of downstream processes involving CCF. This study offers new insights into CCF formation, suggesting a promising direction for further research.

    However, there are some limitations to this study. The enhancer mapping for SASP is outdated, as advancements in Hi-C have significantly developed this area. Therefore, the claimed enhancers of SASP may not be accurate. Additionally, the authors did not address what happens in the later stages of CCF formation in the absence of TPR. If TPR is not required for CCF formation at later stages, it fails to explain the downstream processes at these time points adequately. This suggests that TPR may also have another mechanism of SASP regulation independent of CCF formation.

  6. Version published to 10.1101/2024.04.18.590085v1 on bioRxiv