PIE-1 promotes SUMOylation and activation of HDAC1 during the C. elegans oogenesis

  1. Heesun Kim
  2. Yue-He Ding
  3. Shan Lu
  4. Mei-Qing Zuo
  5. Darryl Conte
  6. Meng-Qiu Dong
  7. Craig C. Mello

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    Summary: In this paper you describe experiments showing that PIE-1 is sumoylated at K68, and that K68 sumoylation plays a role in PIE-1 interaction with HDA-1 and its sumoylation, which leads to its activation. The reviewers found the sumoylation dependence of PIE-1 function in piRNA silencing to be of interest, but raised major issues that need to be addressed. In particular, more mechanistic insights into how sumoylation of PIE-1 at K68 enhances HDA-1 sumoylation and regulation are required.

    This is a co-submission with the manuscript https://www.biorxiv.org/content/10.1101/2020.08.17.254466v2

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Abstract

Germlines shape and balance heredity, integrating and regulating information from both parental and foreign sources. Insights into how the germline handles information have come from the identification of factors that specify or maintain the germline fate. In early C. elegans embryos, the CCCH zinc-finger protein PIE-1 localizes to the germline where it prevents somatic differentiation programs. Here we show that PIE-1 also functions in the meiotic ovary where it becomes SUMOylated and engages the SUMO-conjugating machinery. Using whole-proteome mass spectrometry to detect SUMO-conjugated proteins, we identify HDAC SUMOylation as a target of PIE-1. Our findings suggest that SUMOylation activates HDAC, lowering histone acetylation and enhancing Argonaute-mediated surveillance in the germline.

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  1. eLife

    Reviewer #3:

    In the manuscript by Kim et al., show that, beyond its roles of preventing somatic differentiation in the germline of embryos, Zn-finger protein PIE-1 also functions in the adult germline, where it is both SUMOylated as well as interacts with the SUMO conjugating machinery and promotes SUMOylation of protein targets. They identify HDA-1 as a target of PIE-1-induced SUMOylation. Here too, I find the claims interesting, however data is sometimes missing or does not fully support the claims.

    Main concerns:

    1. A key claim of novelty over previously proposed "glue" functions of SUMO is based on the fact that they find that temporally regulated SUMOylation of a very specific residue in a specific protein is affecting protein activity: The observation that "SUMOylation of HDA-1 only appears to regulate its functions in the adult germline" and not in the embryo together with the finding that "other co-factors such as MEP-1 are SUMOylated more broadly, these findings imply that SUMOylation in the context of these chromatin remodeling complexes, does not merely function as a SUMO-glue (Matunis et al., 2006) but rather has specificity depending on which components of the complex are modified and/or when."

    I find this claim poorly supported by the data. In fact, I find that the data supports that multiple SUMOylations contribute to formation of larger complexes: The His-SUMO IP (Fig 2B) brings down far more un-SUMOylated HDA-1 than SUMOylated. This argues for the presence of large complexes with different factors being SUMOylated and many bringing down unmodified HDA-1. The chromatography experiments (Fig 3B-C) also provide hits that are in complex and not direct interactors. Finally, HDA-1 SUMOylation is indicated to regulate MEP-1 interaction with numerous factors (Fig 3D). If all these factors are in one complex, it is hard to imagine how a single SUMO residue would mediate all of these simultaneously. It is quite likely (and not tested) that loss of HDA-1 SUMOylation leads to (partial?) dissociation of a large complex, rather than loss of individual interactions with the SUMO residue of HDA-1. Unlike claimed by the authors, there is no evidence that the "activity" of HDA-1 is regulated by SUMO modification.

    1. Based on loss of MEP-1/HDA-1 interaction upon pie-1 RNAi and smo-1 RNAi (Fig 4B), the authors conclude that "SUMOylation of PIE-1 promotes the interaction of HDA-1 with MEP-1 in the adult germline".

    The evidence that it is PEI-1 SUMOylation that is affecting MEP-1/HDA-1 interaction is fairly weak. In fact, based on Fig 4A, MEP-1 and HDA-1 interact without expression of PIE-1, and in PIE-1 K68R (sumoylation-deficient), although due to poor labeling of the panel it is not clear whether lane 1 and 4 refer to the WT pie-1 locus without tag or lack of pie-1.

    In 4B the HDA-1 band that is present in L4440 but not in pie-1 or smo-1 RNAi is very faint, and in our experience such weak signal is not linear i.e., bands can disappear or appear depending on the exposure. Importantly, according to the data, seemingly unmodified HDA-1 immunoprecipitated with MEP-1 (Fig 4B). This data contradicts the authors' claim that "These findings suggest that in the adult germline only a small fraction of the HDA-1 protein pool, likely only those molecules that are SUMOylated, can be recruited by MEP-1 for the assembly of a functional NURD complex".

    Furthermore, the fact that pie-1 and smo-1 depletion eliminate the interaction between HDA-1/MEP1 doesn't mean that the SUMOylation of pie-1 specifically is required for the interaction: perhaps un-SUMOylated pie1, and SUMOylation of something else, are both necessary for the interaction. The authors show that MEP-1 is also SUMOylated (Fig3C). When IP-ing GFP-MEP-1, they precipitate all its modified forms and associated factors. One alternative possibility for why smo-1 RNAi abolishes MEP-1/HDA-1 interaction is that MEP-1 SUMOylation is needed for interaction with HDA-1 (independently of pie-1). (On a side note, why are the authors not including MEP-1 SUMOylation in the model?)

    1. On page 13 the authors write: "These findings suggest that SUMOylation of PIE-1 on K68 enhances its ability to activate HDA-1 in the adult germline" and "We have shown that PIE-1 is also expressed in the adult germline where it engages the Krüppel-type zinc finger protein MEP-1 and the SUMO-conjugating machinery and functions to promote the SUMOylation and activation of the type 1 HDAC, HDA-1 (Figure 6)". Activation of HDA-1 is misleading and was never tested. If not performing in vitro assays for HDAC activity, the authors at least need to look at whether pie loss (degron) leads to acetylation of genomic HDA-1 targets and whether it affects HDA-1 (and/or MEP-1) recruitment to these sites. This could be done by ChIP-seq of HDA-1 and H3K9ac in WT and pie-1 degron animals.
  2. eLife

    Reviewer #2:

    In their manuscript, Kim et al address the role of PIE-1 sumoylation during C. elegans oogenesis. The authors favour a model in which sumoylated PIE-1 acts as a sort of E3-like factor 'enhancing' HDA-1 sumoylation. While the results are indeed very interesting, it is unclear to me whether there is enough data to support the author's model. I have list of comments, suggestions, questions, and concerns, which are listed below, which I hope will help the authors strengthen the manuscript:

    Figure 1)

    I) As with the accompanying manuscript, the extremely low level of SUMO modification should be factored in the model.

    II) Is sumoylation also observed in untagged pie-1? As judged by figure 3A, the authors have a very good antibody to test this.

    III) While the authors claim that PIE-1 sumoylation is not observed in embryos, that panel shows a lower exposure than the corresponding one in Adult (as judged by the co-purified unmodified PIE-1::FLAG). A longer exposure and/or more loading would be helpful.

    IV) Their strategy and optimisation for purification of sumoylated proteins is excellent and will be useful for future research (along with other reagents the authors developed here). Is the 10xHis::smo-1 functional? Could this be tested in vitro and/or in vivo?

    V) In vitro PIE-1 sumoylation would be a desirable addition to this figure.

    VI) In addition to germline PIE-1 localisation, it would be interesting to see embryos and PIE-1(K68R).

    VII) MW markers are missing in the blots.

    Figure 2)

    I) The generation of the ubc-9 ts allele is an exceptional tool. Could the authors show SUMO conjugation levels at permissive vs restrictive temperature? Just out of curiosity, is this a fast-acting allele?

    II) The authors mention that gei-17 alleles are viable, could the authors mention any thoughts on why the tm2723 allele is lethal/sterile?

    Figure 3)

    I) Panel C is mentioned in the text in the wrong place. Also in C, what do the authors think about the big increase in MEP-1 sumoylation in the PIE-1(K68R) background?

    II) I have the same comment for panel D as I had for figure 1 comment III: the exposure/loading for the embryo WB seems lower, as judged by the co-purifying, unmodified HDA-1. A positive control for sumoylated protein coming from embryos would be nice.

    III) In general, the model of PIE-1 acting as a SUMO machinery recruiter should be tested with recombinant proteins. Even if compatible with some results in vivo, showing that this is a plausible mechanism in vitro would be extremely helpful and greatly support the authors' claim.

    Figure 4)

    I) The authors make a quantitative comparison of the HDA-1/MEP-1 interaction in the text. I think this is not correct. Even if these have been run in the same gel, this could just be a lower exposure. In this line, the HDA-1 blot in the 'Adult' IP would benefit from a longer exposure to better appreciate what seems a rather small difference between PIE-1 and PIE-1(K68R).

    II) Since there still seems to be interaction between MEP-1 and HDA-1 in the PIE-1(K68R) background, does smo-1(RNAi) or ubc-9(G56R) reduce this further?

    III) In panel B, the LET-418 blot on the right is massively overexposed.

    IV) Once again, in vitro binding experiments to get some indication that the authors' model is plausible would be a great addition.

    Figure 5)

    I) Could the authors make some quantitation of the immunofluorescence data?

    Overall, I think this manuscript proposes a very interesting model and the results support this model, although I am not convinced these are sufficient to strongly back the authors' claims. I would very much like to see a revised version with some in vitro data backing the authors' model.

  3. eLife

    Reviewer #1:

    The evidence that sumoylation of K68 in the PIE-1 zinc finger protein is important for HDA-1 type 1 histone deacetylase association and sumoylation seems reasonable, and, is important because as shown in the co-submitted paper HDA-1 sumoylation leads to its association with MEP-1 and LET-418/NuRD complex thus accelerating H3K9ac deacetylation, and silencing gene expression.

    The evidence that PIE-1 is needed for sumoylation of HDA-1, presumably through association of PIE-1 with the UBC-9 SUMO E2, is reasonable. However, several aspects of the authors' model remain unclear, and there is an absence of biochemical assays to establish the role of sumoylated PIE-1 in HDA-1 sumoylation, and the effects of sumoylation on HDA-1 HDAC activity.

    1. How sumoylation of K68 in PIE1 affects its function was not worked out. Can the deleterious effect of the K68R mutation on PIE-1 function be reversed by generating a SUMO-PIE-1 fusion, as was done for HDA-1 in the co-submitted paper? K68 maps to the N-terminal side of ZF1 in the PIE-1 protein in what appears to be an unstructured region. Does the SUMO residue play a role in the interaction of PIE-1 with HDA-1? Are the zinc fingers required for PIE-1 interaction with HDA-1 or UBC-9? No zinc finger mutations were tested. Does HDA-1 have a SIM that would allow it to interact selectively with sumoylated PIE-1? Another possibility is that the PIE-1 SUMO moiety is important because it interacts with the non-covalent SUMO-binding site on the backside of UBC-9 (Capill and Lima, JMB 369:606, 2007), which might stabilize the interaction. The backside interaction of SUMO with UBC-9 is proposed to promote UBC-9-mediated sumoylation of target proteins with SUMO consensus sites that are directly recognized by UBC-9. In this scenario, SUMO-PIE-1 would in effect be acting as an E3 SUMO ligase for HDA-1 by serving as a recruitment "factor". In this regard, the authors could test biochemically whether recombinant PIE-1 or K68SUMO-PIE-1 stimulates sumoylation of HDA-1 by UBC-9, using recombinant WT and KKRR mutant HDA-1 as substrates. These issues deserve discussion.

    2. What is the SUMO E3 ligase that sumoylates PIE-1? Is it possible that through association with UBC-9, perhaps through its zinc fingers, PIE-1 is sumoylated in cis within a PIE-1/UBC-9 complex?

    3. In many places, including the title, the authors make the claim that PIE-1 promotes sumoylation and activation of HDA-1. While it is clear that PIE-1 does increase sumoylation of HDA-1, in a manner requiring K68, and that H3K9ac levels are decreased as a result, the authors do not provide any direct evidence that this process increases HDA-1 catalytic activity, as is implied in the title and elsewhere. As indicated in the review of the co-submitted paper, this would need to be established by carrying out an HDAC assay on control and sumoylated HDA-1 in vitro. Instead of enzymatic activation, it is possible that the PIE-1 interaction and HDA-1 sumoylation results in relocalization of HDA-1 within the nucleus to facilitate more efficient H3K9ac deacetylation.

  4. eLife

    Summary: In this paper you describe experiments showing that PIE-1 is sumoylated at K68, and that K68 sumoylation plays a role in PIE-1 interaction with HDA-1 and its sumoylation, which leads to its activation. The reviewers found the sumoylation dependence of PIE-1 function in piRNA silencing to be of interest, but raised major issues that need to be addressed. In particular, more mechanistic insights into how sumoylation of PIE-1 at K68 enhances HDA-1 sumoylation and regulation are required.

    This is a co-submission with the manuscript https://www.biorxiv.org/content/10.1101/2020.08.17.254466v2

  5. Version published to 10.1101/2020.08.17.253955v2 on bioRxiv
  6. Version published to 10.1101/2020.08.17.253955v1 on bioRxiv