Drosophila p53 isoforms have overlapping and distinct functions in germline genome integrity and oocyte quality control

  1. Ananya Chakravarti
  2. Heshani N. Thirimanne
  3. Brian R. Calvi

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Abstract

p53 gene family members in humans and other organisms encode a large number of protein isoforms whose functions are largely undefined. Using Drosophila as a model, we find that a p53B isoform is expressed predominantly in the germline where it colocalizes with p53A into subnuclear bodies. It is only p53A, however, that mediates the apoptotic response to ionizing radiation in the germline and soma. In contrast, p53A and p53B both respond to meiotic DNA breaks and are required during oogenesis to prevent persistent germline DNA breaks, an activity that is more crucial when meiotic recombination is defective. We find that in oocytes with persistent DNA breaks p53A is required to activate a meiotic pachytene checkpoint. Our findings indicate that Drosophila p53 isoforms have DNA lesion and cell type-specific functions, with parallels to the functions of mammalian p53 family members in the genotoxic stress response and oocyte quality control.

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  1. eLife

    ###Reviewer #3:

    In this manuscript, Chakravarti and colleagues analyzed the functions of several p53 isoforms in the Drosophila germline. They created novel isoform-specific alleles by CRISPR/Cas9 to untangle the functions of p53A and p53B isoforms. They made use of a Phid-GFP reporter line to follow p53 transcriptional activity. The role of p53 in the development of Drosophila germline has been published several times before with a focus on the silencing of retro-transposons (TEs) and meiotic DNA breaks response (Lu, 2010; Wylie, 2014; Wylie, 2016). Despite this published literature, the authors created novel and very valuable tools, which allowed them to make several novel and interesting observations. My main criticism is that most of these observations remain unexplained and the manuscript feels descriptive as it stands. However, this manuscript has great potential if it could follow up some of these novel observations. Some examples are the following:

    1. In Figure 5C, the authors made the interesting observation that hid-GFP was stronger in region 1 of p53A-B+ than in the wild type p53A+B+. This activity of p53 cannot be explained by meiotic DSBs as previously published, since meiotic DSBs only occur later in region 2. This observation remains unexplained and is not explored further.

    One possibility is that it could relate to transposable elements (TEs) activity in this region. TEs can create DSBs (thus non-meiotic) and p53 has been published to silence TEs in Drosophila (Wylie, 2014; Wylie, 2016). It is also particularly interesting that the silencing of TEs is known to be weakened in this specific region of the germarium even in wild type condition (Dufourt J, NAR, 2013; Theron E, NAR, 2018). Could p53A play a role in silencing TEs in this region when Piwi is downregulated? This would bring novel insights on when and where TEs are silenced in germ cells.

    A transcriptomic analysis of p53A-B+ germ cells could show whether TEs are upregulated in this hid-GFP++ cells. It is probably out of the scope of this manuscript. Another possibility would be to perform FISH for TEs known to be expressed in p53 mutant, such as TAHRE (Wylie, 2016). In addition, do the authors detect DSBs in region 1 in p53A-B+?

    1. On Figure 7 and 8, the authors analyzed the role of p53 in "persistent" meiotic DSBs. I am not convinced that these DSBs are only persistent meiotic DSBs. As discussed by the authors themselves (page 13), the origin of these DSBs could be TEs mobilization. I think it is a very important caveat for their conclusions. Another non-exclusive possibility for DSBs appearing in endoreplicating nurse cells is incomplete replication and associated DNA deletions during repair as shown in (Yarosh and Spradling, GD, 2014).

    To distinguish between these possibilities and strengthen their conclusions, the authors should perform the same experiments in the absence of meiotic DSBs, such as in a meiW68 mutant background (meiW68, p53AB double mutant). meiW68, okra, p53 mutants may be hard to generate but shRNAs against meiW68 are publicly available and effective, while they may also exist for okra or other spindle genes, and could make this combination easier to generate.

    1. The authors showed that p53A and p53B levels are developmentally regulated (Figure 6G): does overexpression of one or both of the isoforms have any phenotype?

    2. I agree with the authors that karyosome defects are part of an array of phenotypes induced by the activation of DNA damage checkpoints. However, I would not equal it to the activation of a pachytene checkpoint and conclude that p53 is part of that checkpoint.

    3. On Figure 7D, in p53A+B-, there seems to be a lot of DNA damages in follicular cells. Is this reproducible?

  2. eLife

    ###Reviewer #2:

    The Drosophila genome encodes multiple p53 isoforms. P53 is an important factor in maintaining genome integrity and having multiple isoforms in flies raises an interesting evolutionary concept because humans have a gene family of p53 members. In this paper, the expression and function of the isoforms is compared in the germ line. There are two significant findings based on investigating these two isoforms. First, the apoptotic response depends on the A form, and both have roles in the response to meiotic DSBs. These results represent a significant and important extensions of previous work from another group that showed p53 suppresses transposon activity.

    With one important exception, the data are solid and support the conclusions. The data regarding the apoptotic response is based on TUNEL and a hid-GFP reporter. This data shows that irradiation induces a response in the mitotic region but not later regions. Conversely, there is a milder induction in the meiotic region (region 2a). Both could be in response to DSBs. But it is amazing that there is no HID induction following IR in these meiotic regions. Thus, there is a satisfying correlation between the apoptosis and HID responses to IR, and both are diminished in the meiotic region.

    The most significant concern with this paper is that conclusions that the p53 isoforms respond to meiotic DNA breaks. Indeed, this is the title of the section starting at the end of pg 7, but there are no experiments which lead to this conclusion. Similarly, the sentence "To determine whether p53A or p53B isoforms responds to meiotic DNA breaks" (pg 8), is followed by an experiment which does not do that (it compares HID expression in different p53 genotypes). The data in the paper are correlations between p53 expression and where DSBs occur in the germarium. Two experiments are needed. First, and most important, hid-GFP expression needs to be analyzed in a mei-W68 mutant. In addition, the germarium should be stained for both HID and gH2AV, the latter being the antibody the authors use in later Figures. It would also be satisfying to see the genotypes in Figure 7 performed in a mei-W68 mutant background, to determine if the persistent DNA damage in the p53 mutants depends on meiotic breaks.

  3. eLife

    ###Reviewer #1:

    In this manuscript Chakravarti et al build on the previous work from the Calvi lab characterizing specific roles for the p53A isoform. In their 2015 paper Zhang et al showed, using isoform specific loss of function mutants, that p53A is primarily responsible for mediating the apoptotic response to ionizing radiation in the soma and that p53B is very lowly expressed in the cell types studied. They speculated that p53B might function in germline specific roles, such as meiotic checkpoints and DNA repair, identified in mammalian p53 studies.

    Here Chakravarti et al, have further characterized the functions of the p53A and B isoforms in Drosophila. In the ovary, p53A mediates the apoptotic response to IR and is also required for meiotic checkpoint activation. p53B is both necessary and sufficient for repair of meiotic breaks in nurse cells but not oocytes. p53B is required for expression of a hid-GFP reporter in region 2a-2b cells which may be related to a loss of p53B detection in p53A/B nuclear bodies at that stage.

    There are no substantive concerns with this manuscript.

    Minor concerns: CRISPR/Cas9 was used to create isoform-specific mutants for both p53A and p53B. RT-PCR was used to show the mutant alleles are isoform specific and that neither disrupts the expression of the others endogenous protein. The RT-PCR assay can only assess the expression of isoforms, not their function as the authors state.

    The authors noted that, even in the absence of IR, there was low level hid-GFP expression in late region 1/early region 2, the point when meiotic DSBs are induced by Mei-W68. Quantitation of hid-GFP expression in the various p53(A+/-,B+/-) mutant backgrounds showed that hid-GFP expression in the absence of IR requires p53 activity and that both isoforms are capable of activating hid-GFP expression. The authors suggest that the increased and earlier expression of hid-GFP seen in the p53A-/p53B+ mutant is due to precocious hyperactivation by p53B unrelated to meiotic breaks which have yet to occur. The authors then seem to contradict themselves saying that p53 reporter construct expression is dependent on Mei-W68, and both isoforms respond to DSBs. Since p53B is capable of precocious activation of at least one p53 target in the absence of p53A expression it is not clear that meiotic breaks themselves directly regulate p53B activity. From the data presented it seems plausible that p53A responding to DSBs might attenuate p53B activity. Quantitation of p53A and p53B levels across oogenesis shows a transient reduction in p53B levels in regions 2a-2b which coincides with the timing of meiotic breaks. Again, it is unclear whether this is a direct response of p53B to meiotic breaks. The authors suggest this change in p53B concentration in the p53A/B body might be due to transient relocalization from the p53A/B body to the nucleoplasm and back but that variation in fluorescence intensity makes it impossible to accurately assess levels in the nucleoplasm to confirm this. While p53B is undetectable in region 2a-2b cells, its presence is required there for expression of hid-GFP, thus translocation from the p53A/B body to fulfill this function is plausible.

    The figures are well done and appropriate to the message, however, in the fluorescent images the high background in the mCh channel makes it difficult to see the true signal and it is often completely lost in the merged images. Perhaps use of a greyscale panel would be more informative.

    In 2019 Park et al, using Gal4/UAS transgenes in a p53 null background concluded that both p53A and p53B mediated the apoptotic response to IR in the Drosophila ovary. I feel the authors adequately addressed this issue in stating that their current results using loss of function, isoform-specific alleles at the endogenous locus better reflects the true physiological response. Thus, I feel their conclusions on the role of p53 in the ovary have more merit.

  4. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 2 of the manuscript.

    This manuscript is in revision at eLife.

    ###Summary:

    The authors have generated new and useful p53 reagents, which they have employed in four functional assays: apoptosis (TUNEL after 40 Gy irradiation (Figures 2-3), transcriptional induction (monitored by hid-GFP (Figures 4-5)), double stranded DNA breaks (DSB) (monitored by gammaH2AV (Figures 7-8)) and activation of pachytene checkpoint (monitored by synaptonemal complex protein C(3)G (Figure 8F-K)).

    The main findings are: 1) the apoptotic response to ionizing radiation (IR) depends on p53A; 2) expression of hid-GFP in region 2a-2b germ cells requires p53B; 3) DSBs occur at higher rates in both the p53A and the p53B mutants; and 4) p53B can repair of meiotic breaks in nurse cells but in not oocytes.

    Despite the generation of high-quality, new reagents, this paper is currently fairly descriptive. Of 8 figures, two show the expression pattern of the tagged p53 isoforms in various parts of the germarium (Figs 1 and 6). Some of the observations based on functional assays remain unexplained and need further experiments, including points 1 and 2 below.

    1. The authors conclude that the p53 isoforms respond to meiotic DNA breaks, but there are no experiments which lead to this conclusion. If the authors want to conclude this, they need (a) to analyze hid-GFP expression a mei-W68 mutant and (b) stain the germarium with both HID and gammaH2AV. The authors should also examine meiotic breaks in p53A+B+, p53A-B-, p53A-B+ and p53A+B- in a background that is also mei-W68 mutant.

    2. The authors are missing a more detailed analysis of the interesting observation that hid-GFP is stronger in region 1 of p53A-B+ than in the wild type p53A+B+. This observation cannot be explained by meiotic DSBs (which occurs in region 2), but the authors do not provide a mechanism. Is this due to transposable elements? The authors need to supply new data to provide a mechanistic understanding of this observation.

    3. The authors are encouraged to provide better data to support the conclusion that the DNA damage phenotypes of p53 and okra mutants are comparable. The images in Figs. 7, 8B and B' are not sufficient to assess this. The authors could quantify the number of gammaH2AV foci or intensity (rather than measure the number of positive cells). Related to this, it is surprising that p53 mutants lack the DV defects seen in okra mutants, particularly since defects in DSB repair should cause nondisjunction. Okra mutants are sterile. The authors should comment upon the fertility of p53 mutants.

    4. Some experiments have only 2 biological replicates (Figs 4 and 8K). Figs 7 and 8 have "2-3 replicates". The authors need to state specifically for each experiment how many replicates were scored. Ideally, they should have at least 3 replicates for each experiment or explain why that is not necessary.

  5. Version published to 10.1101/2020.07.21.214692 on bioRxiv