Identification of epigenetic modulators as determinants of nuclear size and shape

  1. Andria C Schibler
  2. Predrag Jevtic
  3. Gianluca Pegoraro
  4. Daniel L Levy
  5. Tom Misteli

  • Curated by eLife

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    eLife Assessment:

    In this manuscript the authors describe targeted, imaging-based RNAi screens to identify novel modulators of nuclear size and shape — two traits that are diagnostic and prognostic for many human diseases including cancers. The work provides novel insights into how and what dictates nuclear morphology, further decoupling key different components of lamins, chromatin, and the nuclear envelope, but there are some notable concerns regarding the scoring approach applied in the screen and hit validation. The authors also provide new evidence that lamin A may directly bind to (modified) histone H3 and how histone H3 disease mutations impact nuclear shape; this aspect of the manuscript would benefit from a more thorough analysis.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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Abstract

The shape and size of the human cell nucleus is highly variable among cell types and tissues. Changes in nuclear morphology are associated with disease, including cancer, as well as with premature and normal aging. Despite the very fundamental nature of nuclear morphology, the cellular factors that determine nuclear shape and size are not well understood. To identify regulators of nuclear architecture in a systematic and unbiased fashion, we performed a high-throughput imaging-based siRNA screen targeting 867 nuclear proteins including chromatin-associated proteins, epigenetic regulators, and nuclear envelope components. Using multiple morphometric parameters, and eliminating cell cycle effectors, we identified a set of novel determinants of nuclear size and shape. Interestingly, most identified factors altered nuclear morphology without affecting the levels of lamin proteins, which are known prominent regulators of nuclear shape. In contrast, a major group of nuclear shape regulators were modifiers of repressive heterochromatin. Biochemical and molecular analysis uncovered a direct physical interaction of histone H3 with lamin A mediated via combinatorial histone modifications. Furthermore, disease-causing lamin A mutations that result in disruption of nuclear shape inhibited lamin A-histone H3 interactions. Oncogenic histone H3.3 mutants defective for H3K27 methylation resulted in nuclear morphology abnormalities. Altogether, our results represent a systematic exploration of cellular factors involved in determining nuclear morphology and they identify the interaction of lamin A with histone H3 as an important contributor to nuclear morphology in human cells.

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  1. Version published to 10.7554/elife.80653 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    The screening effort has revealed a number of interesting and novel suggestions of new modulators of nuclear appearance that are exciting and have the potential to be of value to the field.

    We appreciate the reviewer’s view that identification of new modulators of nuclear morphology is exciting and of value to the field.

    Major Points:

    1. The discussion of the screen hits and prior knowledge key to their interpretation is lacking. For example, the authors only report on the purported localization of the hits without an unbiased analysis of their function(s). As a sole example, multiple members of the condensin complex are hits in Fig.1 while multiple members of the cohesin complex are hits in Fig. 2 - but there are many more factors worthy of further discussion. Moreover, the authors need to provide more information on the data used to assign the localization of the hits and how rigorous these assignments may be. For example, multiple CHMP proteins (ESCRTs) are listed - indeed CHMP4B is the highest scoring hit in Fig.1 - but this protein does not reside at the nuclear envelope at steady-state; rather, it is specifically recruited at mitotic exit to drive nuclear envelope sealing. Moreover, there are many hits for which there is prior published evidence of a connection to nuclear shape or size that are ignored: examples include BANF1, CHMP7, Nup155 (and likely far more that I am not aware of). This is a missed opportunity to put the findings into context and to provide a more mechanistic interpretation of the type of effects that lead to the observed changes in nuclear appearance. For example, there is already hints as to whether the effects occur as a mitotic exit defect versus an interphase defect, but conceptually this is not addressed.

    We appreciate this important point. We find that one of the major challenges in presentation of screening results is to provide detailed information on all interesting hits within the length limits of a manuscript! To provide a more comprehensive picture, we have now performed pathway analysis using STRING to display protein interaction networks to more comprehensively classify hits and groups of hits (Figures S7 and S8). We find highly connected regions in the network corresponding to condensin and histone modifiers in fibroblast hits altering nuclear shape. In contrast, MCF10AT hits showed increased connectivity with nucleoporin proteins. Fibroblast hits displaying an increase in nuclear size identified multiple nucleoporins and MCF10AT hit analysis identified components of DNA replication. We have added these findings to Supplementary Figures 7 and 8 and discuss them on page 16. Also, as requested, we added more than 20 new references and additional information on previously identified functions of some hits discussed in the text on p. 22-24.

    1. Validation of the screen is lacking. There appears to be no evidence that the authors validated the initial screen hits by addition siRNA experiments in which the levels of the knock-down could be assessed. As an example: do nucleoporin hits decrease in their abundance at the nuclear envelope in these conditions? This validation is absolutely essential.

    As requested, we now include in Tables S6A-C, data from independent validation experiments in which we selected the primary hits and validated them using an independent set of siRNAs with distinct chemistry and target sequences. Additionally, we demonstrate efficient knockdown capabilities for 8 targets in Supplementary Figure 9 with knockdown levels for most siRNAs of at least 60%. We find no strong relationship between knockdown efficiency and the extent of the observed phenotype (compare Figure S9 and Figure S10).

    1. Differences in cell type - the authors' interpretation that a lack of overlap in the hits across cell types reveals that there are fundamentally cell type-specific mechanisms at play is a stretch. This could also reflect a lack of robustness in the screen, which should be addressed by directly testing the knock-down of the hits from one cell line in the other. Even if this approach reinforces the cell type specificity, the differences in the biology beyond the nucleus itself - an obvious example being the mechanical state of the cell - organization of the cytoskeleton, adhesions, etc that influence forces exerted on the nucleus are different rather than the nuclear response is different. These caveats needs to be explicitly acknowledged.

    As requested, we have now performed side by side experiments between both cell lines to directly compare a subset of nuclear morphology hits in parallel. They are shown in Supplementary Figure 10. We find a number of hits display strong nuclear shape abnormalities in either fibroblasts or MCF10AT cells but not both, with the exception of LMNA, which confirms our screen data. In addition, we compared the hits from our screen with previously published reports of other factors which regulate nuclear morphology to further strengthen our findings. We mention these results on p. 16. Despite these results, we have now toned down our statements regarding cell-type specificity of individual hits considering the small number of cell lines analyzed and the possible cellular factors which could contribute to cell-type specific differences.

    1. There are major issues with the interpretation of the presented biochemistry. For example, the basis for the supposed effect of monomer/dimer state of lamin is confusing and likely misinterpreted. It is well established that GST imposes dimerization on proteins expressed as GST fusions independent of cysteines. Any effect of DDT would have to manifest through some other mechanism (disulfides between the lamin domains - assumedly what the authors are thinking). Further, GST will impose dimerization of lamin A and lamin C in the co-incubation experiments. It is therefore entirely expected that if lamin A binds H3 and lamin C does not that the mixed dimers will bind H3 with lower affinity. Critically, this does not, however, address how full-length lamin C influences binding of lamin A to H3 in vivo. Last, how an effect of lamin C on lamin A would manifest through a disulfide bond in the nucleus, which has a reducing environment, is entirely unclear.

    We directly tested the possibility that GST causes artifactual dimerization of lamins by mutating cysteines to alanine in GST-lamin and assessing their effect on histone binding experiments. We show the results in Supplementary Figure 14E. If the observed binding were artifactually due to GST-mediated dimerization, we should not expect an effect of the cystine mutants on histone binding. We find, however, that the C522A mutation in lamin A results in increased binding of H3 in the presence of lamin C, demonstrating that the observed effects are not due to GST dimerization. We discuss these results on p. 18 and p. 19.

    We agree with the referee that it will be exceptionally challenging to determine the in-vivo relevance of disulfide bonds, not knowing what the precise environment of the nucleus is. Given these caveats, we have now toned down this point and discuss the limitations of these findings in more detail on p. 19, 23, 24, and 25.

    1. It is important for the authors to address the concept of nuclear size changes versus changes in the nuclear to cell volume ratio – biologically these could be quite different conditions, but obviously these cannot be distinguished by measuring nuclear volume alone. Addressing this experimentally would be best (to provide more depth to the size measurements).

    This is an important point. As requested, we now clearly indicate on p. 23 that we are measuring nuclear area using nuclear cross-sections as a proxy for nuclear size rather than nuclear to cell volume ratio. We have found in our imaging studies over the past two decades that measuring cell volumes is exquisitely challenging and often highly inaccurate. A major challenge in these approaches is the correct identification of cell boundaries and this is particularly challenging in a high-throughput setting since cell volume measurements require z-stacks that greatly complicates the imaging and quantitative analysis and increases the complexity of this kind of analysis of the millions of cells analyzed in a screen. Ultimately, measurements of cell volume for adherent cells will only be estimates (see for example PMID 28622449). We now clearly indicate this limitation of our approach and discuss on p. 15 and 23 previous studies measuring nuclear size and cell volume ratio measurements and how it compares to measuring nuclear area alone. We have also added several references on this topic on p. 15 and 23.

    1. There are important caveats to the approach of using the nuclear area as proxy measurement for nuclear size, most prominently that it is highly responsive to changes in nuclear height that can occur for a multitude of reasons (increased height = small radius and decreased height = larger radius), particularly given the different cell types. This needs to be acknowledged directly.

    Along the lines of point 5 and as requested, we now more clearly acknowledge on p. 23 these caveats due to our screening method of measuring nuclear area as a proxy for nuclear size. Nuclear cross-sectional area has been experimentally shown to be a good proxy for nuclear size in many systems (see PMID 31085625). For this reason, and because quantifying nuclear size from z-stacks would have greatly complicated the imaging and quantitative analysis, we chose to use nuclear cross-sectional area as our metric for nuclear size. In looking through our data, we did not find any significant differences in nuclear height between the two cell lines used or amongst hits and non-hits. With respect to the issue of different cell types, our analysis focused on RNAi knockdowns that altered nuclear morphology in a given cell line and we did not compare cell lines against each other. Separate analyses were performed for each cell line, so possible differences in nuclear height between the different cell lines used should not affect our analysis. We now discuss these issues on p. 23.

    1. What is the evidence that the H3 effects manifest through lamins rather than directly?

    We apologize for not being clear. We did not mean to intend to state that H3 acts via lamins. We do find that H3 physically interacts with lamins and that H3.3 mutants (K9M, K27M, and K36M) result in nuclear morphology defects. We now also show in the new Figure S17 that H3.3 mutants slightly affect lamin levels. However, as pointed out by the reviewer, these observations do not categorically rule out non-lamin related mechanisms and we now make it clear in our discussion on p. 20 that the effect of H3 may either be mediated via lamins or independently.

    1. Context is needed for the "methyl-methyl" histone states described as being the highest binders in the peptide array experiments. Are these states commonly found? Where in the genome? Does this match any DamID data? Again - more depth of investigation is required.

    This is a good point. Unfortunately, to our knowledge there is currently no ChIP-seq human genome map of di-methyl modifications on histone tails available. We were unable to generate or procure the individual dually methylated peptides and methyl-methyl H3 antibodies are not available and we are thus not able to perform quantitative binding assays. However, to begin to address this issue, we now provide in a new Supplementary Table 8 quantitative data of binding intensities. Given these limitations, we have now toned the claims regarding the methyl-binding sites.

    1. That oncohistones induce changes in nuclear shape or size does not mean that this is related to the mechanism in cancer. Also - how over-expression of H3 without its obligate partner H4 could disrupt the cell or an assessment of the extent of the oncohistone incorporation into chromatin achieved in these experiments makes it challenging to interpret.

    We agree and did not intend to imply that the oncogenic function of the histone mutants involves changes in nuclear morphology. We now clearly state so on p. 25 and we also mention the caveat of the overexpression experiment.

    1. Throughout the manuscript it would be helpful to the reader if the author would provide at minimum a brief statement on the previously identified functions of the hits that are explicitly discussed beyond their localization (membrane versus chromatin). References would also be helpful (for example, again - what is the evidence that SLC27A3 resides at the nuclear envelope?).

    As requested, we added more than 20 new references and now provide additional information and previously identified functions of many of the hits mentioned in the text.

  3. eLife

    eLife Assessment:

    In this manuscript the authors describe targeted, imaging-based RNAi screens to identify novel modulators of nuclear size and shape — two traits that are diagnostic and prognostic for many human diseases including cancers. The work provides novel insights into how and what dictates nuclear morphology, further decoupling key different components of lamins, chromatin, and the nuclear envelope, but there are some notable concerns regarding the scoring approach applied in the screen and hit validation. The authors also provide new evidence that lamin A may directly bind to (modified) histone H3 and how histone H3 disease mutations impact nuclear shape; this aspect of the manuscript would benefit from a more thorough analysis.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    Summary: In this manuscript the authors report an RNAi screen to identify proteins that, when depleted, alter nuclear shape and/or nuclear size. They demonstrate that the changes cannot be solely attributed to changes in the expression of nuclear lamins. Many of the hits are factors that impinge on histone modifications and chromatin biology. Comparing hits between fibroblasts and an epithelial cell type (MCF10) demonstrated relatively little overlap. The authors then relate their observations to a potential direct interaction between lamin A and histone H3 that, using histone peptide arrays, may be modulated by the methylation status. Last, the authors find that over-expression of some histone H3 variants/oncohistones alters nuclear appearance.

    Overall assessment: The screening effort has revealed a number of interesting and novel suggestions of new modulators of nuclear appearance that are exciting and have the potential to be of value to the field. Unfortunately, the remainder of the manuscript is largely descriptive and rather superficial; perhaps most importantly validation experiments to rigorously confirm the screen "hits" are lacking. There are also concerns about the interpretation of biochemical experiments into lamin A-histone H3 binding although there are also some promising hints into the histone modification-dependence of lamin A binding to H3 that, if more fully investigated, would be an important contribution.

    Major Points:

    1. The discussion of the screen hits and prior knowledge key to their interpretation is lacking. For example, the authors only report on the purported localization of the hits without an unbiased analysis of their function(s). As a sole example, multiple members of the condensin complex are hits in Fig.1 while multiple members of the cohesin complex are hits in Fig. 2 - but there are many more factors worthy of further discussion. Moreover, the authors need to provide more information on the data used to assign the localization of the hits and how rigorous these assignments may be. For example, multiple CHMP proteins (ESCRTs) are listed - indeed CHMP4B is the highest scoring hit in Fig.1 - but this protein does not reside at the nuclear envelope at steady-state; rather, it is specifically recruited at mitotic exit to drive nuclear envelope sealing. Moreover, there are many hits for which there is prior published evidence of a connection to nuclear shape or size that are ignored: examples include BANF1, CHMP7, Nup155 (and likely far more that I am not aware of). This is a missed opportunity to put the findings into context and to provide a more mechanistic interpretation of the type of effects that lead to the observed changes in nuclear appearance. For example, there is already hints as to whether the effects occur as a mitotic exit defect versus an interphase defect, but conceptually this is not addressed.

    2. Validation of the screen is lacking. There appears to be no evidence that the authors validated the initial screen hits by addition siRNA experiments in which the levels of the knock-down could be assessed. As an example: do nucleoporin hits decrease in their abundance at the nuclear envelope in these conditions? This validation is absolutely essential.

    3. Differences in cell type - the authors' interpretation that a lack of overlap in the hits across cell types reveals that there are fundamentally cell type-specific mechanisms at play is a stretch. This could also reflect a lack of robustness in the screen, which should be addressed by directly testing the knock-down of the hits from one cell line in the other. Even if this approach reinforces the cell type specificity, the differences in the biology beyond the nucleus itself - an obvious example being the mechanical state of the cell - organization of the cytoskeleton, adhesions, etc that influence forces exerted on the nucleus are different rather than the nuclear response is different. These caveats needs to be explicitly acknowledged.

    4. There are major issues with the interpretation of the presented biochemistry. For example, the basis for the supposed effect of monomer/dimer state of lamin is confusing and likely misinterpreted. It is well established that GST imposes dimerization on proteins expressed as GST fusions independent of cysteines. Any effect of DDT would have to manifest through some other mechanism (disulfides between the lamin domains - assumedly what the authors are thinking). Further, GST will impose dimerization of lamin A and lamin C in the co-incubation experiments. It is therefore entirely expected that if lamin A binds H3 and lamin C does not that the mixed dimers will bind H3 with lower affinity. Critically, this does not, however, address how full-length lamin C influences binding of lamin A to H3 in vivo. Last, how an effect of lamin C on lamin A would manifest through a disulfide bond in the nucleus, which has a reducing environment, is entirely unclear.

    5. It is important for the authors to address the concept of nuclear size changes versus changes in the nuclear to cell volume ratio - biologically these could be quite different conditions, but obviously these cannot be distinguished by measuring nuclear volume alone. Addressing this experimentally would be best (to provide more depth to the size measurements).

    6. There are important caveats to the approach of using the nuclear area as proxy measurement for nuclear size, most prominently that it is highly responsive to changes in nuclear height that can occur for a multitude of reasons (increased height = small radius and decreased height = larger radius), particularly given the different cell types. This needs to be acknowledged directly.

    7. What is the evidence that the H3 effects manifest through lamins rather than directly?

    8. Context is needed for the "methyl-methyl" histone states described as being the highest binders in the peptide array experiments. Are these states commonly found? Where in the genome? Does this match any DamID data? Again - more depth of investigation is required.

    9. That oncohistones induce changes in nuclear shape or size does not mean that this is related to the mechanism in cancer. Also - how over-expression of H3 without its obligate partner H4 could disrupt the cell or an assessment of the extent of the oncohistone incorporation into chromatin achieved in these experiments makes it challenging to interpret.

    10. Throughout the manuscript it would be helpful to the reader if the author would provide at minimum a brief statement on the previously identified functions of the hits that are explicitly discussed beyond their localization (membrane versus chromatin). References would also be helpful (for example, again - what is the evidence that SLC27A3 resides at the nuclear envelope?).

  5. eLife

    Reviewer #2 (Public Review):

    This manuscript reports a large gene screen of hundreds of genes using siRNA knockdown for changes in nuclear shape via roundness and size across lamin, chromatin, and nuclear envelope proteins. Using this approach this paper reports that changes in lamin A or B1 levels does not account for most of the abnormal nuclear morphology hits. Instead, many of the hits are either related to nuclear envelopes proteins or chromatin modifying proteins, with a specific group responsible for repressive histone modifications. The screen is also done in a second cell type MCF 10A where interestingly abnormal nuclear morphology do not overlap with initial screen in fibroblasts. The paper then goes on to detail how the C-terminus of lamin A interacts/binds with the histone tails/modifications. Finally, the paper reports that modifications in histone H 3.3, which are associated with disease, also cause abnormal morphology. This work encompasses many interesting findings to the field of nuclear biology and provides novel insights into how and what dictates nuclear morphology further decoupling key different components of lamins, chromatin, and nuclear envelope. However, the manuscript's current form undermines or complicates the data for the reader by graphing and reporting one z score instead of nuclear roundness and size, and this also occurs for other assays/figures. These graphs provide no way to compare variance between replicates, in replicates, or across different siRNA KD constructs - as no graphs have error bars or individual data points. Providing a clearer understanding to the reader of the core data would greatly increase the paper's impact and the readers' understanding of the large amount of very interesting and valuable data collected.

  6. eLife

    Reviewer #3 (Public Review):

    Nuclear shape and size have long been characterized and can indicate and influence cell fates. The present study starts with a knockdown screen of size and shape, adds some information on lamins known to influence size and shape, proceeds to focus on 'subtle' modulators that are often epigenetic factors, then provides in vitro pulldown and array studies that support histone-lamin interactions, and concludes with further such evidence from one small final cell study. Some concerns temper enthusiasm. I found it important that they restricted analyses "To eliminate hits due to cell death or altered cell-cycle behavior, we excluded any hits with a cell number z-score of less than -2." Some mention of this in the abstract seems important. Secondly, The histone-H3 mutation effects on nuclear morphology in Fig.6 are especially important, but it is unclear whether the histone intensities are uniform or enriched in places with LaminA, nor what happens to LaminA levels or localization.

  7. Version published to 10.1101/2022.05.28.493845v1 on bioRxiv