The role of RNA in the maintenance of chromatin domains as revealed by antibody-mediated proximity labelling coupled to mass spectrometry

  1. Rupam Choudhury
  2. Anuroop Venkateswaran Venkatasubramani
  3. Jie Hua
  4. Marco Borsò
  5. Celeste Franconi
  6. Sarah Kinkley
  7. Ignasi Forné
  8. Axel Imhof

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Abstract

Eukaryotic chromatin is organized into functional domains, that are characterized by distinct proteomic compositions and specific nuclear positions. In contrast to cellular organelles surrounded by lipid membranes, the composition of distinct chromatin domains is rather ill described and highly dynamic. To gain molecular insight into these domains and explore their composition, we developed an antibody-based proximity biotinylation method targeting the RNA and proteins constituents. The method that we termed antibody-mediated proximity labelling coupled to mass spectrometry (AMPL-MS) does not require the expression of fusion proteins and therefore constitutes a versatile and very sensitive method to characterize the composition of chromatin domains based on specific signature proteins or histone modifications. To demonstrate the utility of our approach we used AMPL-MS to characterize the molecular features of the chromocenter as well as the chromosome territory containing the hyperactive X chromosome in Drosophila . This analysis identified a number of known RNA-binding proteins in proximity of the hyperactive X and the centromere, supporting the accuracy of our method. In addition, it enabled us to characterize the role of RNA in the formation of these nuclear bodies. Furthermore, our method identified a new set of RNA molecules associated with the Drosophila centromere. Characterization of these novel molecules suggested the formation of R-loops in centromeres, which we validated using a novel probe for R-loops in Drosophila . Taken together, AMPL-MS improves the selectivity and specificity of proximity ligation allowing for novel discoveries of weak protein–RNA interactions in biologically diverse domains.

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  1. Version published to 10.7554/elife.95718 on eLife
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    The authors will submit a complete point-by-point response to the reviewer's comments when submitting a fully revised version of the manuscript

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    Referee #4

    Evidence, reproducibility and clarity

    This is a short report featuring an innovative proximity labeling approach to the identification of proteins enriched in distinct types of chromatin domains. The domains compared are centromeric heterochromatin and X-linked hyperactive chromatin in Drosophila cells. These are relatively well-described domains, thus serving as an excellent test for the targeting of biotinylation in the permeabilized nucleus via interaction of specific antibodies with ProteinA-Apex2 provided exogenously. In parallel with the signature chromatin proteins CID or MSL2 as baits, the authors also target proteins in proximity to specific histone tail PTMs. Taking the work one step further, they compare the recovery of proteins +/- pretreatment of nuclei with RNase. They conclude that in each case selective interactions are specifically lost with pre-treatment of RNase.

    Major comment:

    As mentioned above, the approach is innovative and raises the possibility of a simpler MS method to identify protein-protein interactions. The RNase result is also provocative. However, in each case the specificity of potentially novel results are not explored further. Thus, the work is of interest but clearly still preliminary.

    Significance

    Did the authors dig deeper into novel interactions without obtaining convincing validation? Did they conclude that the MS approach is worth pursuing further or not? Admittedly the RNase result is difficult to follow up, but additional discussion of prior related work as well as consideration of future experiments would help improve the manuscript.

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript entitled 'The role of RNA in the maintenance of chromatin domains as revealed by antibody mediated proximity labelling coupled to mass spectrometry' by Choudhury et al. describe a new method, which they termed AMPL-MS (Antibody mediated proximity labelling mass spectrometry). The technique is based on proximity labelling but uses antibodies instead of fusion proteins. They use this method to characterize chromatin domains containing specific signature proteins or histone modifications and focus on the composition of chromocenter as well as the chromosome territory containing the hyperactive X-chromosome in Drosophila. Last but not least they include data that show that RNA is involved in maintaining the integrity of chromatin domains by RNAse treatment and mass spec analysis. The technique works well and the results are very clear. I therefore expect that, in the right hands, it is very reproducible.

    There are a few points that the authors may want to address:

    1. Title 'The' role of RNA in the maintenance of chromatin domains as..., seems too much of a statement. The title is therefore an overstatement that needs to be fixed.
    2. Figure 1 In Figure 1 the authors show very convincingly that the methods works well in their hands. They report on 172 proteins that localized in proximity to CID containing centromeric chromatin but do not provide the list of proteins as far as I can tell. Especially the RNA binders should be named.
    3. Figure 2 Using the hyperactive X is very clever when addressing RNA function but it should be stated in the discussion that there may be certain aspects that are specific to the male x and that is impossible to discriminate general and specific effects uncovered by this method.
    4. Figure 3 The authors should state more clearly the new findings of this figure since it is not fully obvious from its current representation.
    5. Figure 4 These are certainly interesting data but the authors remain in the very descriptive state. This is fine for a methods paper but then, the authors should hypothesize more on what the results mean. Are certain RNA dependent factors specific or general and they then recruit a specific set of factors that fall off upon RNAse treatment as a secondary effect or because they bind RNA directly. I feel like there may be more information that they authors got get out of there data than what they currently provide.
    6. Discussion The authors state: 'While we have not identified the RNAs responsible for the formation of theses domains, we clearly observe that they do confer specificity for the domains as we observe very little overlap in the factors lost from the corresponding domains (Fig 4h). the 'specificity' is hard to determine since factors bound to these regions are different, and therefore different factors will fall off, regardless of whether the RBP are specific unless the RNA is involved in recruiting the factors specifically, which the authors have not shown. Therefore, this result is suggestive and interesting but the statement is too strong and not backed by their results.

    Significance

    Overall, this is an interesting method that has been used in the past to identify protein modifications with high quality antibodies available. The authors show here that the method can also be used to different nuclear proteins and detect changes in protein complex composition. As it is it is primarily a methods paper, and for that the results are very clear. Gain of new info is not large but it is a useful technique to continue research on this subjectand is a nice start of many new avenues into how RNA effects chromatin,

    My expertises are in epigenetics, chromatin biology, RNA and Drosophila genetics

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    Referee #2

    Evidence, reproducibility and clarity

    This manuscript by Choudhury et al. describes a new method for antibody-mediated proximity labeling and applies it in the cell nucleus. In short, nuclei are isolated, fix and permeabilized, proteins are labeled with primary antibodies, a bacterially expressed/purified protein-A-APEX2 fusion protein is added, conventional H2O2/biotin phenol labeling of proximate protein is performed, proteins are un-crosslinked and biotin-affinity captured for MS analysis. The application to nuclear proteins and results seems appropriate. The method is highly similar to and more complicated than prior methods as described in more detail below. I would focus the impact of this paper towards its biological results and not the novelty of the methods used.

    Prior methods that effectively accomplish the same outcome (fixed cells/tissues, antibodies and proximity labeling for AP-MS) have been published before. Perhaps most recently it was reinvented as the so called BAR method in PMID 29256494. That paper was cited here but incorrectly as BirA-related, which it is not. Of course that prior manuscript itself ignored prior methods from years back (2008, 2012, 2014, 2015, PMID 18495923, 22936677, 24706754, 25829300) using the same approaches of antibody targeted peroxidase for the same purposes of proximity labeling.

    This method seems a somewhat Rube Golderbergian approach to antibody-mediated proximity labeling, which has been performed previously in multiple reports. APEX/2was developed to function inside of living cells since HRP does not. The value of doing the proximity labeling in living cells was either to capture protein associations over time, as with BioID/TurboID, or to get snapshots of protein associations in living cells with APEX/2. HRP does however function quite well for proximity labeling outside of cells, or in fixed/permeabilized cells, as has been demonstrated in the prior methods/papers that are referenced above. Replacing commercially available secondary antibodies fused to HRP with homemade protein-A-fused to APEX2 seems counterintuitive and/or unnecessary.

    Could the authors explain the mechanisms that underly the reported enhanced sensitivity of AMPL-MS compared to conventional APEX2 in living cells. Is there something about the nuclear isolation that reduces interfering background, the loss of small soluble molecules in the nucleus after isolation and/or permeabilization that enhance the proximity labeling, penetration issues with the biotin-phenol in living cells, and/or something else?

    There seems to be the use of various controls based on the figures and legends, but they are not clearly described in the results or methods.

    All MS results should be provided, preferably in an Excel file format.

    Significance

    This manuscript by Choudhury et al. describes a new method for antibody-mediated proximity labeling and applies it in the cell nucleus. In short, nuclei are isolated, fix and permeabilized, proteins are labeled with primary antibodies, a bacterially expressed/purified protein-A-APEX2 fusion protein is added, conventional H2O2/biotin phenol labeling of proximate protein is performed, proteins are un-crosslinked and biotin-affinity captured for MS analysis. The application to nuclear proteins and results seems appropriate. The method is highly similar to and more complicated than prior methods as described in more detail below. I would focus the impact of this paper towards its biological results and not the novelty of the methods used.

    Prior methods that effectively accomplish the same outcome (fixed cells/tissues, antibodies and proximity labeling for AP-MS) have been published before. Perhaps most recently it was reinvented as the so called BAR method in PMID 29256494. That paper was cited here but incorrectly as BirA-related, which it is not. Of course that prior manuscript itself ignored prior methods from years back (2008, 2012, 2014, 2015, PMID 18495923, 22936677, 24706754, 25829300) using the same approaches of antibody targeted peroxidase for the same purposes of proximity labeling.

    This work may be of interest to investigators studying the nuclear proteins/structures to which the APML-MS was applied.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    This manuscript describes the application of a new variation of proximity (biotin) labelling (antibody mediated proximity labelling coupled to mass spectrometry, AMPL-MS). Combining protein- or histone variant-specific antibodies with a APEX2-proteinA fusion protein, they characterise the proteome of nuclear subdomains and demonstrate that RNA is important for the integrity of two tested domains, the Drosophila chromocenter and the chromosomal territory of the hyperactive X chromosome.

    Major comments:

    The vast majority the experimental results, statistical analysis, and conclusions drawn by the authors appear sound and are described in way that should allow reproduction (however, see my comments below for some suggestions for minor improvements). The authors rigorously test their method, using the Drosophila chromodomain as 'playground', before applying it to other chromosomal areas and histone variants/modifications. Besides providing proteomes of the targeted nuclear subcompartments, they show that RNase treatment of the cells radically changes the proteome(s) and conclude a role for RNA in the integrity of the corresponding compartments. This is shown by immunofluorescence staining as well as proteomic analysis of the biotinylated proteins. The images in figure 4b (and to lesser extent 4c) show an increased intensity and more diffuse labelling. Can the authors exclude that RNase treatment simply leads to an increase in accessibility for the biotin-phenol, hence a visibly higher biotinylation? Along these lines, have the authors maybe observed an increase in overall labelling/pulldown efficiency or for biotinylated proteins in their proteomic data?

    Minor comments:

    1. In figures 1a and 4a (as well as in the Methods section), the authors use the term 'biotin-tyramide' as labelling agent, but in the main text and figure legends 'biotin-phenol' is used. For clarity, only one term should be used.
    2. Figure 2a shows a magnified cell/nucleus in the last column. To what cells do the magnifications in this last column refer to? Maybe these cells could be boxed in the second last column?
    3. In figures 4b + c:, the figure legend mentions the individual rows as '(I)' and '(II)' but no such label seen in the corresponding panel(s).
    4. The Quantification method for co-localization (e.g. 1c and 2b) is insufficiently described to the reader (reference simply relates to Fiji package). What module/script within the Fiji package has been used?
    5. The RNase treatment is not described at all in the methods section or the supplementary information and should be added.
    6. The sentence on page 6 ('As expected, neither the targeted signature factor or proteins that mainly interact with them protein-protein interactions such as MSL1,3 and MOF for MSL2 or Cenp-C for Cid are not affected by RNAase treatment') should be rephrased as it is not comprehensible in the current form.

    Significance

    The findings in this manuscript advance our portfolio of proximity labelling techniques although this advancement is not a major step forward. As the authors state themselves, antibody-based proximity labelling has already been introduced, even in the context of chromosomal proteomes (e.g. Gan et al., 2022; https://doi.org/10.1016/j.gpb.2021.09.003). One major technical advance is the finding that modifications or protein variants can now reliably be targeted for proximity labelling, using their method. Furthermore, the number of cells that are required for proximity labelling and detection of biotinylated proteins could be significantly reduced compared to previous approaches (although this might simply be due to the use of a more advanced proximity labelling enzyme). I should state her that as I am not an expert in the field of chromatin domains, I cannot be certain if the proteomes and changes of proteomes the authors report are providing a significant increase in our knowledge on these domains, especially related to their individual functions.

  7. Version published to 10.1101/2023.10.16.562458v1 on bioRxiv