lncRNA H19/Let7b/EZH2 axis regulates somatic cell senescence

  1. Manali Potnis
  2. Justin Do
  3. Olivia El Naggar
  4. Eishi Noguchi
  5. Christian Sell

  • Curated by eLife

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    eLife Assessment

    In this manuscript, Sell et al., investigate the role of the long non-coding RNA H19 in regulating cellular senescence. Using several cell models they identify upstream and downstream effectors of H19 including let-7 and EZH2. The advances in this work include the identification of a specific cascade of factors connecting H19, senescence and the actions of rapamycin.

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Abstract

Long non-coding RNAs (lncRNAs) regulate diverse cellular processes and are associated with many age-associated diseases. However, the function of lncRNAs in cellular senescence remains largely unknown. Here we characterize the role of lncRNA H19 in senescence. We show that H19 levels decline as cells undergo senescence, and depletion of H19 results in premature senescence. We find that repression of H19 is triggered by the loss of CTCF and prolonged activation of p53 as part of the senescence pathway. Mechanistically, the loss of H19 drives senescence via increased let7b mediated targeting of EZH2. We further demonstrate that H19 is required for senescence inhibition by the mTOR inhibitor rapamycin, where it maintains lncRNA H19 levels throughout the cellular lifespan and thus prevents the reduction of EZH2 that would otherwise lead to cellular senescence. Therefore, lncRNA H19 is crucial in maintaining the balance between sustained cell growth and the onset of senescence.

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  1. eLife

    eLife Assessment

    In this manuscript, Sell et al., investigate the role of the long non-coding RNA H19 in regulating cellular senescence. Using several cell models they identify upstream and downstream effectors of H19 including let-7 and EZH2. The advances in this work include the identification of a specific cascade of factors connecting H19, senescence and the actions of rapamycin.

  2. eLife

    Reviewer #1 (Public Review):

    This is a brief set of experiments that tells a nice story that is relevant to a very important area of biology, namely senescence. The authors identify a role for lncRNA H19 in senescence and delve into the upstream and downstream factors that could describe the phenomenon. They identify CTCF and p53 as upstream regulators of H19 in senescing cells and propose that sponging of let-7 could be a contributing factor to H19's effects via altered regulation of EZH2.

    The work is backed up by strong data in cell models. However, the work could benefit from additional mechanistic data to support the most important conclusions. For example, does H19 sponge let-7 in these cells? What are the relative levels of expression of H19 compared to let-7 in these cells? Is the let-7 binding site on H19 required for the effects of H19? And does let-7 directly regulate EZH2 in these cells? Can a direct role for H19 in affecting EZH2 be ruled out in these cells?

  3. eLife

    Reviewer #2 (Public Review):

    The study aims to characterize the role of lncRNA H19 in senescence and proposes a mechanism involving CTCF and the activation of p53. The authors suggest that H19 loss induces let7b-mediated repression of EZH2, which is a critical component in the regulation of senescence-associated genes. Additionally, the authors state that H19 is required for inhibition of senescence by the mTOR inhibitor rapamycin.

    The experiments appear to be performed to a high standard, and the individual observations, and conclusions about the importance of the individual players in senescence appear solid. For example, the authors convincingly show that H19 decreases in expression in aged cells/tissues and that its knockdown leads to entry into senescence. These results are consistent with recent studies in other systems (e.g., ref 38). Also, the knockdown of CTCF convincingly leads to senescence. However, these observations are largely not very surprising/novel. The premise of the manuscript is a connection between these components into a particular "axis" that regulates entry into senescence. This connection between the different regulators studied (H19, CTCF, EZH2, p53), and in particular, their specificity, which is key to the proposed "axis" remains insufficiently supported, and many of the results, unfortunately, appear to be over-interpreted.

    Major comments

    1. In Figure 1, the authors claim that H19 levels are reduced during aging in vitro and in vivo and that H19 levels are maintained by rapamycin treatment. To state the connection between H19 and rapamycin and its relation to aging, there is a need to show what happens in "young" cells treated with rapamycin.

    Furthermore, the authors state that H19 "is essential for the inhibitory effect of rapamycin on cellular senescence". There doesn't appear to be sufficient evidence to support such a claim; additional data emphasizing the direct connection between H19 and rapamycin is needed - e.g., show that in H19-null cells rapamycin does not affect senescence.

    2. CTCF is a general regulator involved in various cellular processes and supporting progression through the cell cycle; therefore, its perturbation can lead to global effects on cell health that are not necessarily related to H19. The data shown in figure 2 is insufficient to indicate a direct correlation between CTCF and H19. This will require showing that mutating specifically the CTCF binding sites near H19 affects senescence.

    The same applies to the connection between H19 and let-7b shown in Figure 5. It is not very surprising that let-7b, a general antagonist of proliferation, positively regulates senescence. Here as well, the direct connection to H19 is weak. Can the authors rescue the cells that enter senescence following H19 depletion by H19 expression? If so - is this rescue capacity lost when let-7 sites are mutated? Is it possible to rescue by expressing an artificial let-7 sponge instead of H19? Otherwise, let-7b could very well be another factor related to senescence and/or regulated, but not the main mediator of the effects of H19, or part of an axis that includes H19, as proposed in the manuscript.

    3. In figures 2d,3f,5i/j the authors present only representative tracks and regions from CUT&Tag-experiments, and its not clear to what extent these changes are significant when considering genome-wide data, replicates etc., and so these data are uninterpretable. This is important, as these panels are used as evidence for specific connections between members of the axis. The authors should provide a statistical test for all the regions in the genome, based on replicates, and show that these changes are significant to use these data to support their model. Otherwise, the specific connection between CTCF and H19 remains weak, and the specific change in p53 regulation of CTCF in the context of senescence is not convincing. In any case, the number of replicates and the QC of the data should be presented, and the data should be made available to the reviewers.

    4. The authors state in the Discussion that the mechanism that lead to decreased H19 expression as part of the senescence program consists of two phases: an acute response driven by p53 activation and a prolonged response dictated by the loss of CTCF. There doesn't appear to be enough evidence to support this claim, as the individual experiments don't measure any such bi-phasic phenomena.

  4. ASAPbio crowd review

    Review coordinated via ASAPbio’s crowd preprint review

    This review reflects comments and contributions by Luciana Gallo, Lauren Gonzalez, Claudia Molina, Arthur Molines, Srimeenakshi Sankaranarayanan and Sanjeev Sharma. Review synthesized by Iratxe Puebla.

    The manuscript studies the role of the long-coding RNA lncRNA H19 in cellular senescence. The results show that H19 levels decline as cells undergo senescence and repression of H19 is triggered by the loss of CTCF and prolonged activation of p53. The loss of H19 leads to increased let7b-mediated targeting of EZH2. The mTOR inhibitor rapamycin maintains lncRNA H19 levels throughout the cellular lifespan preventing reduction of EZH2 and cellular senescence.

    The reviewers found the methodology appropriate but raised some comments and suggestions about the paper as outlined below:

    Introduction ‘H19 is a highly conserved, maternally expressed imprinted gene and encodes a 2.3 kb long non-coding RNA (lncRNA). It is located immediately downstream of the neighboring gene IGF2.’ - An additional reference to the expression pattern/levels of lncRNA H19 across 'normal' tissues/developmental stages would be useful to provide immediate insight into the contexts where H19 is important and note the conditions where its levels are altered.

    ‘To characterize the role of H19 in the cellular senescence of somatic cells, we examined H19 expression during replicative senescence of human cardiac fibroblasts’ - The data on changes in expression of H19 with age/culture time is very interesting. Suggest providing some comments on the choice of experimental systems for each experiment and why HCF cells were used to study replicative senescence while other experiments were completed in skin samples.

    Figure 1

    • Figure 1a - Please indicate in the legend how far apart or what are the passage numbers for 'early' and 'late' passages for the cell culture experiments. Is the reduction in H19 gradual or does it sharply decrease after a certain number of passages? What biological meaning would either of these observations have and how does it relate to mouse data in vivo?
    • Supplementary Figure 1 shows a sharp drop between PD 20 and PD 50. Would it be possible to provide a finer analysis of H19 levels across many cell passages?
    • Figure 1b - Recommend using the same normalization in a) and b). In a) levels are normalized to the first condition "early" while in b) levels are normalized to the second condition "old".
    • Figures 1d and g - Please provide further information on how Cumulative population doublings were measured and clarification for the numbers on the Y axis.
    • ‘decreased the lifespan of cells (Figure 1d; Figure 1-figure supplement 1c)’ - Figure 1d measures cells' doubling time, not lifespan. If lifespan is being inferred from doubling time, please provide some clarification on how this is being done. There are fewer cells after 15 days but it does not mean that cells are dying, it could be that they are growing slower. Please also provide details for the methodology followed to obtain the data in this panel.

    Figure 2

    • ‘CTCF mRNA and protein levels decreased in the late passage cells (Figure 2a and b), and CTCF knockdown in early passage cells induced premature senescence characterized by increased SA-β-gal staining and reduction in proliferation (Figure 2-figure supplement 2a). In contrast, treatment with rapamycin mitigated CTCF depletion, which is consistent with the effect of rapamycin maintaining H19 levels (Figure 2a and b). Furthermore, the regulatory link between CTCF and H19 is supported by decreased H19 expression in CTCF-targeted cells (Figure 2c).’ - CTCF knockdown and rapamycin treatment can affect many pathways, recommend toning down this conclusion. In Supplemental Figure 2a, the % of positive cells in the siNeg condition is significantly higher than in Figure 1e (close to 50% in Sup Fig 2a vs 30 % in Fig 1e). Recommend providing some comments on the variability of the control value as that level of variability can confound the conclusions. For example, the siCTCF condition is lower than the siNeg control condition when compared with the value from Sup Fig 2a but not when compared with the value from Fig 1e.
    • Figure 2d - Remove "presentation last saved just now" from the panel.

    ‘a stress-dependent downregulation of CTCF through proteasomal degradation of CTCF protein in endothelial cells (51)’ - The paper cited here discusses epithelial cells, should the reference to endothelial cells be updated?

    Figure 3 - Please provide further clarification regarding acute stress or prolonged activation of p53. What are the timescales? How do these relate to replicative senescence seen with aging or as cells at late passages?

    ‘Together these results confirm that activation of p53 is responsible for the downregulation of H19 as part of DNA damage response’ - Please provide further clarification regarding the reference to DNA damage. Is this an inference from the statement about "activation of p53 is crucial for establishing senescence as part of DDR"? p53, like CTCF and mTOR, can play different roles.

    ‘Given the mounting evidence suggesting the role of lncRNA H19 as a competing endogenous RNA (ceRNA) or miRNA sponge (60–62), we speculated that H19 might mediate the senescence program by regulating miRNA availability. To determine which miRNAs are directly regulated by lncRNA H19 during senescence, we evaluated miRNA expression profiles in control and H19 targeted cells (Figure 4a).’ - Can some further clarification be provided for this claim, if H19 is acting as a miRNA sponge, it wouldn't affect its overall levels, but rather its ability to bind its target genest? Based on the data presented, the link between let7b and H19 appears to be more related to let7b expression than sequestration. Consider removing the fragment or revising it to clarify the mechanistic link drawn between H19 and let7b. To show that H19 is acting as a sponge in this system, it may be necessary to mutate the complementary sequence and check whether let7b's activity increases (i.e. its target genes are down-regulated).

    ‘Among the top miRNAs upregulated in H19 depleted cells were members of the let7 family; specifically, let7b expression was significantly upregulated (Figure 4b’ - Suggest adding some more information about the other miRNAs that are affected.

    Figure 4f ‘Senescence-associated secretory’ - Please clarify why SERPINE mRNA level is considered instead of IL-6 as in Figure 1f.

    ‘suggests the loss of EH2 results in a general decrease in PRC2 activity’ - should EH2 read EZH2?

    Figure 5 - What happens to CDKN2A levels when H19 is depleted or overexpressed? Can the H3Kme3 antibody binding data be supported with expression data for CDKN2A? It may be relevant to see whether it follows the expectation that loss of H19 reduces EZH2 expression and increases p16 expression.

    Figure 6 - Please provide some brief clarification for what the solid and dashed lines represent in the model.

    ‘More importantly, prolonged treatment with mTOR inhibitor rapamycin maintains lncRNA H19 levels by preventing the loss of CTCF expression and activation of p53, thus preventing the induction of senescence.’ - There is a question as to whether the experiments presented support this statement, suggest reframing the fragment. The strongest mechanistic experiments in the study are those regarding let7b, because they use the mimic to "rescue" its function.

    Supplementary Figure 1d - It is nice to see authors tested 2 different siRNAs for H19 and these showed the same effect in Panel d. Can some discussion be provided for why overexpression of H19 leads to an increase in senescence markers and reduced proliferation.The outcomes of siRNA experiments may not sufficiently support the correlation between H19 levels and senescence induction. This is an example where both excess H19 and reduced levels of H19 have the same effect and it is a very important result. Would it be possible to titrate the expression of H19 to achieve different levels of overexpression and then analyze senescence markers under these conditions? It may also be possible to generate a siRNA-resistant overexpression construct to rescue the effects seen with siRNA-mediated depletion of H19.

    Supplementary Figure 5 - Recommend updating the presentation to more clearly highlight the decrease in binding as mentioned in the main text.

    Methods

    • ‘10g of plasmid DNA was transfected’ - should this read 10 micrograms?
    • ‘ΔΔCT method’ - Please clarify the control for calculating relative mRNA levels.
    • ‘Cells were incubated with EdU stain (100mM Tris (pH8.5), 1mM CuSO4, 1.25 μM Azide Fluor 488, and 50mM ascorbic acid) at room temperature for 30 mins. Cells were washed with PBS twice and imaged using EVOS FL Auto microscope (Thermo Fisher)’ - Please report the duration that the cells were incubated with EdU in culture before the cells were fixed and EdU incorporated in the DNA was stained.
  5. Version published to 10.1101/2022.07.07.499142v2 on bioRxiv
  6. Version published to 10.1101/2022.07.07.499142v1 on bioRxiv