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.
Introduction
The p53 protein is best known as a tumor suppressor that plays a central role in the response to DNA damage and other types of stress (Lane and Crawford 1979; Linzer and Levine 1979; Levine 2020). In response to stress, p53 mostly acts as a homotetrameric transcription factor to induce gene expression that elicits cell cycle arrest, apoptosis, or autophagy, although it also has other non-transcription factor activities (Levine 2020). It is now clear, however, that p53 regulates a growing list of other biological processes, including metabolism, stem cell division, immunity, and DNA repair (Levine 2019). Vertebrate genomes encode two other p53 paralogs, p63 and p73, which also have diverse functions in stress response and development (Jost et al. 1997; Yang et al. 1998; Dotsch et al. 2010; Candi et al. 2014). Adding to this complexity, each of these three p53 family encode a large number of isoforms which can form homo- or heterocomplexes, both within and among the different gene paralogs (Fujita et al. 2009; Aoubala et al. 2011; Joruiz and Bourdon 2016; Anbarasan and Bourdon 2019; Fujita 2019). However, the function of only a small subset of these isoform complexes have been defined. In this study, we use the p53 gene in Drosophila as a simplified genetic system to examine the function of p53 isoforms and find that they have critical overlapping and distinct functions during oogenesis.
The Drosophila melanogaster genome has a single p53 family member (Ingaramo et al. 2018). Similar to human p53 (TP53), it has a C terminal oligomerization domain (OD), a central DNA binding domain (DBD) and an N terminal transcriptional activation domain (TAD), and functions as a tetrameric transcription factor (Jin et al. 2000; Ollmann et al. 2000). This single p53 gene expresses four mRNAs that encode three different protein isoforms (Figure 1A) (Ingaramo et al. 2018). A 44 kD p53A protein isoform was the first to be identified and is the most well characterized (Brodsky et al. 2000; Jin et al. 2000). Later RNA-Seq and other approaches revealed that alternative promoter usage and RNA splicing results in a 56 kD p53B protein isoform, which differs from p53A by an 110 amino acid longer N-terminal TAD that is encoded by a unique p53B 5’ exon (Roy et al. 2010; Ingaramo et al. 2018) (Figure 1A). Because the p53A isoform differs from p53B by a shorter N terminus, p53A is also known as ΔNp53 (Dichtel-Danjoy et al. 2013). Another short p53E mRNA isoform is predicted to encode a protein of 38 kD that contains the DNA binding domain but lacks the longer N-terminal TADs of p53A and p53B (Roy et al. 2010; Zhang et al. 2015) (Figure 1A).
Like its human ortholog, Drosophila p53 regulates apoptosis in response to genotoxic stress, but is now known to mediate other stress responses and developmental processes (Brodsky et al. 2000; Sogame et al. 2003; Wells et al. 2006; Dichtel-Danjoy et al. 2013; de la Cova et al. 2014; Napoletano et al. 2017; Tasnim and Kelleher 2018; Zhou 2019). To promote apoptosis, p53 protein directly induces transcription of several proapoptotic genes at one locus called H99 (Brodsky et al. 2000; Sogame et al. 2003; Zhou 2019). These early analyses of p53 function in apoptosis focused on the p53A isoform because the others had yet to be discovered. Using BAC rescue transgenes that were mutant for either p53A or p53B, we previously showed that in larval tissues it is the shorter p53A, and not p53B, that is both necessary and sufficient for the apoptotic response to DNA damage caused by ionizing radiation (Zhang et al. 2015). In contrast, when each isoform was overexpressed, p53B was much more potent than p53A at inducing proapoptotic gene transcription and the programmed cell death response, likely because of the longer p53B TAD (Dichtel-Danjoy et al. 2013; Zhang et al. 2015). Other evidence suggests that p53B may regulate tissue regeneration and has a redundant function with p53A to regulate autophagy in response to oxidative stress (Dichtel-Danjoy et al. 2013; Robin et al. 2019). It is largely unknown, however, why the Drosophila genome encodes a separate p53B isoform and what its array of functions are.
The p53 gene family is ancient with orthologs found in the genomes of multiple eukaryotes, including single-celled Choanozoans, which are thought to be the ancestors of multicellular animals (Rutkowski et al. 2010). Evidence suggests that the ancestral function of the p53 gene family was that of a p63-like protein in the germline, with later evolution of p53 tumor suppressor functions in the soma (Gebel et al. 2017; Levine 2020). In mammals, p63 mediates a meiotic pachytene checkpoint arrest in response to DNA damage or chromosome defects, and also induces apoptosis of a large number of oocytes with persistent defects, thereby enforcing an oocyte quality control (Di Giacomo et al. 2005; Suh et al. 2006; Gebel et al. 2017; Rinaldi et al. 2017; Rinaldi et al. 2020). It has been shown that in the Drosophila germline p53 regulates stem cell divisions, responds to programmed meiotic DNA breaks, and represses mobile elements (Lu et al. 2010; Wylie et al. 2014; Wylie et al. 2016). In this study, we have uncovered that the Drosophila p53A and p53B isoforms have redundant and distinct functions during oogenesis to protect genome integrity and mediate the meiotic pachytene checkpoint arrest, with parallels to the germline function of mammalian p53 family members in oocyte quality control.
Results
The p53B isoform is more highly expressed in the germline
Our previous results indicated that p53B does not mediate the apoptotic response to radiation in larval imaginal discs and brains (Zhang et al. 2015). One explanation for this lack of function was that p53B protein is expressed at very low levels in those somatic tissues (Zhang et al. 2015). Given the ancestral function of p53, we considered the possibility that p53B may be expressed and function in the germline. To address this question, we evaluated p53 isoform expression and function in the ovary. During Drosophila oogenesis, egg chambers migrate down a structure called the ovariole as they mature through 14 morphological stages (Figure 1B) (King 1970). Each egg chamber is composed of an oocyte and 15 sister germline nurse cells, all interconnected by intercellular bridges (Figure 1B) (Spradling 1993). The nurse cells become highly polyploid through repeated G / S endocycles during stages 1-10 of oogenesis, which facilitates their biosynthesis of large amounts of maternal RNA and protein that are deposited into the oocyte. The germline cells are surrounded by an epithelial sheet of somatic follicle cells that divide mitotically up until stage 6, and then undergo three endocycles from stages 7-10 (Calvi et al. 1998; Deng et al. 2001; Jia et al. 2015). Both germline and somatic follicle cell progenitors are continuously produced by germline and somatic stem cells that reside in a structure at the tip of the ovariole known as the germarium (King 1970; Drummond-Barbosa 2019).
To evaluate p53A and p53B expression during oogenesis, we used fly strains transformed with different p53 genomic BAC transgenes in which the p53 isoforms are tagged on their unique N-termini. In one strain, GFP is fused to p53A (GFP-p53A), while in another strain mCherry is fused to p53B (mCh-p53B), with each expressed under control of their normal regulatory regions in these genomic BACs (Figure 1C, D) (Zhang et al. 2015). To enhance detection, we immunolabeled these strains with antibodies that recognize GFP and mCherry. Immunofluorescent analysis of the somatic follicle cells revealed that GFP-p53A localized to nuclear bodies, ranging in size from ~0.25-1 μm, often in close proximity to the DAPI bright pericentric heterochromatin (Figure 1E,G). The expression of mCh-p53B, however, was only rarely detected in somatic follicle cells (< 1/ 50,000 cells) (Figure 1F,H, Figure S1). Thus, similar to our previous results in larval tissues, p53A is expressed at much higher levels than p53B in somatic cells (Zhang et al. 2015). In contrast, both GFP-p53A and mCh-p53B bodies were detected in all germline cells. Early stage nurse cells had one to a few p53 bodies, whereas later stage nurse cells had more bodies that were regionally distributed in the nucleus (Figure 1E, F, I, J). This dynamic pattern suggests that p53 bodies, like some other nuclear bodies, may associate with the polytene chromatin fibers that become dispersed in these nurse cells after stage 4 of oogenesis (Dej and Spradling 1999; Liu et al. 2006; White et al. 2007; Liu et al. 2009). The oocyte nucleus also had both GFP-p53A and Ch-p53B nuclear bodies, often appearing as one large (~1μm) and several smaller (~0.25μm) bodies (Figure 1K, L). In addition to distinct nuclear bodies, there were low levels of GFP-p53A and mCh-p53B dispersed throughout the nuclei of nurse cells and the oocyte.
We examined females with both GFP-p53A and mCh-p53B to address if they colocalize to the same nuclear bodies. In some cells co-expression of GFP-p53A reduced the expression of mCh-p53B, perhaps a manifestation of a protein trans-degradation effect that we had described previously (Zhang et al. 2014). Nonetheless, the results indicated that GFP-p53A and mCh-p53B colocalize to the same subnuclear bodies of both nurse cells and oocytes, although the ratio of these two isoforms differed somewhat among bodies (Fig 1M-P). mCh-p53B also co-localized with GFP-p53A in those rare follicle cells that expressed mCh-p53B (Figure S1B, C). Examination of the testis indicated that GFP-p53A and mCh-p53B are also expressed and localized to nuclear bodies in the male germline (Figure S2A-B’) (Mauri et al. 2008; Monk et al. 2012). Altogether, these results indicated that while the p53A isoform is expressed in both somatic and germline cells, the p53B isoform is primarily expressed in the germline.
p53A is necessary and sufficient for the apoptotic response to ionizing radiation in somatic follicle cells
We next asked which of the p53 isoforms mediate the apoptotic response to DNA damage in the ovary. We had previously addressed this question in larval imaginal discs and brains using mutant BAC rescue transgenes (Zhang et al. 2015). For this study, we used CRISPR/Cas9 to create isoform-specific mutants at the endogenous p53 locus. Since our previous study indicated that the short p53E is a repressor, we focused on making mutants of p53A and p53B isoforms to distinguish their different activities. The resulting p53A2.3 allele is a 23bp deletion and 7bp insertion within the unique p53A 5’ exon (Figure 2A, Figure S3A). This deletion extends downstream into the first p53A intron removing both p53A coding sequence and first RNA splicing donor site (Figure 2A, Figure S3A) (Robin et al. 2019). This coding sequence and splice donor site are shared with p53C mRNA, which is predicted to encode a p53 protein isoform that is identical to that encoded by p53A mRNA (Figure 1A). Therefore, p53A2.3 disrupts both p53A and p53C protein coding. The p53B41.5 allele is a 14bp deletion plus 1bp insertion in the unique second coding exon of p53B, removing p53B coding sequence and creating a frameshift with a stop codon soon afterward (Figure 2B, Figure S3A). We had previously shown that p53A mRNA structure is perturbed and p53A protein is undetectable in homozygous p53A2.3 animals, whereas p53B mRNA is still expressed (Figure S3B) (Robin et al. 2019). Conversely, RT-PCR indicated that in the p53B41.5 strain the p53A isoform is still expressed (Figure S3B). Thus, p53A2.3 and p53B41.5 alleles are specific to each isoform and do not disrupt the function of the other isoform. This is in contrast to the p535A-1-4 null allele which deletes the common C-terminus of all the isoforms. To be clear about which of these isoforms are expressed from different p53 alleles, we will annotate wild type p53+ as (A+B+), the p535A-1-4 null allele as (A-B-), the p53A specific mutant p53A2.3 as (A-B+) and the p53B specific mutant p53B41.5 as (A+B-) (Figure 2A, B).
To determine which p53 isoforms mediate the apoptotic response to DNA damage, we irradiated adult females from these strains with 40 Gray (Gy) of ionizing radiation (IR) and evaluated cell death four hours later by TUNEL. We focused on the follicle cells in the mitotic cycle up until stage 6 because we had previously shown that endocycling follicle cells in later stage egg chambers repress the p53 apoptotic response to DNA damage (Mehrotra et al. 2008; Hassel et al. 2014; Zhang et al. 2014; Qi and Calvi 2016). In wildtype p53+ (A+B+) ovaries that express both isoforms, approximately 30% of mitotic cycling follicle cells were TUNEL positive, whereas the p53A2.3 (A-B+) mutant had very few TUNEL-positive follicle cells (~1%), which was not significantly different than irradiated p535A-1-4 (A-B-) null or unirradiated controls (Figure 2C-F, H). In contrast, the p53B41.5 (A+B-) mutant strain had 30% TUNEL-positive follicle cells, a fraction similar to that of wild type (Figure 2D, G, H). These results suggested that p53A, but not p53B, is required for the apoptotic response to DNA damage. A possible caveat, however, is that both the p53A2.3 and p53B41.5 alleles also delete part of a non-coding RNA of unknown function (CR46089), which overlaps the 5’ end of p53 and is transcribed in the opposite direction (Roy et al. 2010; Thurmond et al. 2019). Given that this noncoding RNA is disrupted in both alleles, its disruption cannot explain the impaired apoptosis specifically in the p53A2.3 allele. Moreover, similar results were obtained when p53A2.3 or p53B41.5 alleles were transheterozygous to the p535A-1-4 null allele that does not delete portions of this non-coding RNA. These results strongly suggest that mutation of non-coding RNA CR46089, or possible cryptic mutations on the p53A2.3 and p53B41.5 chromosomes, are not contributing to the apoptotic phenotypes. Thus, the p53A protein isoform is both necessary and sufficient for the apoptotic response to IR in somatic ovarian follicle cells.
p53A is necessary and sufficient for the apoptotic response to ionizing radiation in the female germline
The low level of expression of p53B in somatic tissues may explain why it does not mediate the apoptotic response. We wondered, therefore, whether p53B participates in the apoptotic response in the germline where it is more highly expressed. Given that endocycling nurse cells and the meiotic oocyte repress p53 apoptosis, we analyzed the apoptotic response of mitotically-dividing germline cells during early oogenesis in the germarium (Mehrotra et al. 2008; Hassel et al. 2014; Zhang et al. 2014; Qi and Calvi 2016). At the anterior tip of the germarium, the germline stem cells (GSCs) reside in a niche and divide asymmetrically into a GSC and cystoblast (CB) (Figure 3A) (Hinnant et al. 2020). This cystoblast and its daughter cells undergo four rounds of divisions with incomplete cytokinesis as they migrate posteriorly through germarium region 1, finally resulting in an interconnected 16-cell germline cyst. (Figure 3A) (Drummond-Barbosa 2019; Hinnant et al. 2020). In region 2a, multiple cells in the cyst initiate meiotic breaks and synaptonemal complex formation, but only one cell is eventually specified to be the oocyte, with the 15 other cells of the cyst destined to become nurse cells that enter a polyploid endocycle by germarium region 3 (stage 1 of oogenesis) (Figure 3A). To evaluate which cells in the germarium express GFP-p53A and mCh-p53B, we co-labeled with an antibody against the fly adducin protein ortholog called Hu-li tai shao (Hts), which labels a spherical cytoplasmic spectrosome in GSCs, and a cytoskeletal structure called the fusome that branches through the ring canals that connect the 16 cells of a germline cyst (Figure 3A) (Lin et al. 1994). Similar to later stages of oogenesis, both GFP-p53A and mCh-p53B were expressed in GSCs and their daughter germline cells of the germarium where the p53 isoforms colocalized in distinct p53 nuclear bodies (Figure 3B, C).
To determine which p53 isoforms are required for IR-induced germline apoptosis, we irradiated wild type and p53 mutant females with 40 Gy of gamma rays and TUNEL labeled their ovaries four hours later. In wild type p53+ (A+B+) controls, there were an average of ~13 TUNEL-positive germline cells in region 1 of each germarium (Figure 3D,E,I). Although earlier GSCs and later meiotic cells express both p53 isoforms, they did not label with TUNEL (Figure 3E). In p535A-1-4 (A-B-) null ovaries, only ~1 germline cell per germarium was TUNEL-positive in region 1, a number similar to that in unirradiated controls, indicating that most of the germline cell death four hours after IR is p53-dependent (Figure 3 D, F, I). Similar to p535A-1-4 null, the p53A2.3 (A-B+) mutant also had ~1 TUNEL-positive germline cell per germarium (Figure 3G, I). In contrast, the p53B41.5 (A+B-) mutant ovaries had ~12 TUNEL positive cells / germarium, a number similar to that in wild type and significantly greater than that in p53 null and p53A2.3 mutants (Figure 3H, I). These results suggest that, despite p53B expression, it is the p53A isoform that is necessary and sufficient for the apoptotic response to IR in the germline.
To further evaluate p53 isoform function, we determined whether p53A or p53B protein isoforms induce transcription of proapoptotic genes after IR. Previous studies showed that among the proapoptotic p53 target genes, the gene hid plays a prominent role for inducing germline apoptosis in response to DNA damage (Xing et al. 2015; Park et al. 2019). We therefore used a GFP promoter-reporter for hid (hid-GFP), which contains the hid promoter but not coding region, together with GFP antibody labeling to assay p53 transcription factor activity (Figure 4A) (Tanaka-Matakatsu et al. 2009). Although GSCs did not apoptose in p53+ (A+B+), they had high levels of hid-GFP expression after IR (Figure 4C) (Wylie et al. 2014; Xing et al. 2015; Ma et al. 2016). In contrast, IR did not induce high levels of hid-GFP in meiotic cells in region 2, suggesting that in these cells apoptosis is repressed upstream of proapoptotic gene expression (Figure 4C). Similar to the results for TUNEL, expression of the hid-GFP reporter in region 1 cystocytes was induced by IR in p53+(A+B+) wildtype and p53B41.5 (A+B-) mutants, but not in p535A-1-4 (A-B-) null or p53A2.3 (A-B+) mutants (Figure 4B-J). Together, these results indicate that, similar to the soma, p53A is necessary and sufficient for induction of proapoptotic gene expression and the apoptotic response to IR in the germline.
Both p53A and p53B isoforms respond to meiotic DNA breaks
The results indicated that although both p53A and p53B protein isoforms are expressed in the early germline, it is p53A that mediates the apoptotic response to IR in these cells. This result left unanswered what function p53B protein may have in the germline. A clue came when we noticed that there was a low level of hid-GFP expression beginning in late region 1 / early region 2a of the germarium even in the absence of IR (Figure 4B,D,F,H,J). This result is similar to one from the Abrams lab who found that in the absence of exogenous stress germline cells have low levels of expression of a promoter-reporter for another p53 target gene at the H99 locus, reaper (rpr-GFP), in germarium region 2, a time in oogenesis when meiotic DNA breaks are induced by the fly orthologue of Spo11 (Mei-W68) (Mehrotra and McKim 2006; Lu et al. 2010). They showed that this meiotic rpr-GFP expression was undetectable in either mei-W68 or p53 null mutants, indicating that it was dependent on both meiotic DNA breaks and p53 (Lu et al. 2010).
To determine whether the p53A or p53B isoform responds to meiotic DNA breaks, we compared hid-GFP expression among the different p53 alleles. To do this, we increased the exposure times for the unirradiated germaria from Figure 4, and replotted the quantification of their fluorescence intensity (Figure 5A-E). This clearly showed that in p53+(A+B+) wildtype germaria there is a low level of hid-GFP expression in all cells of a germline cyst in the absence of IR beginning in late region 1 / early region 2a, which was undetectable in the p535A-1-4 (A-B-) null mutant (Figure 5A,B, E). Thus, this hid-GFP expression in the absence of IR is dependent on p53 activity, similar to rpr-GFP that reports p53 activity in response to meiotic DNA breaks, (Lu 2010). In the p53B41.5 (A+B-) mutant, hid-GFP expression was decreased to about 50% of wild type levels (Figure 5D,E). Given that hid-GFP expression was undetectable in the p53 null, this result suggests that both p53A and p53B activate hid-GFP. Surprisingly, the p53A2.3(A-B+) mutant had higher levels of hid-GFP expression than wild type, and expression occurred earlier in oogenesis in region 1, including GSCs (Figure 5C, E). This result suggests that in the absence of the p53A isoform, the p53B isoform hyperactivates precocious expression of the hid-GFP reporter. This earlier hid-GFP expression in the p53A2.3 (A-B+) mutant is not a response to meiotic DNA breaks, which are not induced until region 2a (Mehrotra and McKim 2006). Similar results were obtained when the isoform specific mutants were in trans to the p535A-1-4 null allele, indicating the results are not due to cryptic second-site mutations (data not shown). Altogether, these results suggest that both p53A and p53B are required for wild type levels of hid-GFP expression in the absence of IR. Given that p53 reporter expression in these cells is dependent on Mei-W68 (Spo11), these results further suggest that both isoforms respond to meiotic DNA breaks.
Dynamic p53B isoform abundance in p53 bodies correlates with meiotic DNA breaks
To investigate the relationship of p53 isoforms to meiotic DNA breaks further, we examined p53 isoform localization in the early germline. The level of GFP-p53A in p53 bodies was comparable among germline cells in all regions of the germarium, including GSC, dividing cystocytes in region 1, and during early stages of meiosis in regions 2a-2b (Figure 6A, A’). Ch-p53B was also abundant in p53 bodies in GSCs and most region 1 cystocytes (Figure 6B, B’). In contrast, the levels of p53B in the p53 bodies decreased at the onset of meiosis in late region 1 / early region 2, remained low in regions 2a-2b, and then increased again in most cells in late region 2b / early region 3 (Figure 6B, B’). Quantification of GFP-p53A and Ch-p53B levels within the same p53 bodies of GFP-p53A / Ch-p53B females confirmed that although Ch-p53B and GFP-p53A intensity in the bodies is approximately equal in region 1, p53B levels decrease to ~41% that of p53A in regions 2a-2b, and then increase again to levels comparable to p53A in regions 2b-3 (Figure 6C-G, Figure S4). This transient reduction in p53B levels in regions 2a-3 represents only ~24 hours of oogenesis, suggesting that it may be caused by relocalization of p53B protein from the p53 body to the nucleoplasm and then back again, rather than degradation and resynthesis of p53B protein (King 1970; Morris and Spradling 2011). The low magnitude and high variance of the nucleoplasmic fluorescence, however, precluded a determination as to whether the decrease of mCh-p53B in bodies is associated with a commensurate increase in the nucleoplasm (Figure 6E, S4 C-C’”). The transient decrease of p53B in bodies coincided with the known timing of meiotic DNA break induction in region 2a followed by subsequent DNA break repair by region 3. Moreover, p53B is required during this period to induce wild type levels of hid-GFP. These results suggest, therefore, that there may be a functional relationship between dynamic p53B localization and its response to meiotic DNA breaks.
p53A and p53B mutants have persistent germline DNA damage
The hid-GFP results suggested that both p53A and p53B isoforms respond to meiotic DNA breaks. To investigate whether p53 isoforms regulate germline DNA breaks, we labeled ovaries with antibodies against phosphorylated histone variant H2AV (g-H2AV), which marks sites of DNA damage and repair, evident as distinct nuclear DNA repair foci (Madigan et al. 2002; Lake et al. 2013). It has been shown that labeling for g-H2AV detects repair foci at meiotic DNA breaks beginning in region 2a (Jang et al. 2003; Mehrotra and McKim 2006; Lake et al. 2013). Mei-W68 induces breaks in most cells of the 16-cell cyst, but g-H2AV repair foci are most abundant in four cells, one of which will become the oocyte while the others are destined to become nurse cells (Carpenter 1975; Jang et al. 2003; Mehrotra and McKim 2006; Lake et al. 2013; Hughes et al. 2018). Consistent with these previous reports, we observed that ovaries from wild type females had four cells per cyst with intense g-H2AV labeling, first appearing at the onset of meiotic recombination in germarium region 2a, decreasing in region 2b, and then undetectable in 97% of oocytes by germarium region 3 (oogenesis stage 1), a time when most meiotic DNA breaks have been repaired (Figure 7A, A’).
In contrast, females homozygous mutant for the p535A-1-4 (A-B-) null allele had more than four germline cells per cyst with strong g-H2AV labeling (Figure 7B). This phenotype is similar to that of DNA repair mutants that increase the steady state number of unrepaired g-H2Av-positive breaks, and thereby the number of cells per cyst that label strongly for g-H2AV (Figure 7B) (Mehrotra and McKim 2006; Wei et al. 2019). Also similar to known DNA repair mutants, repair foci in p535A-1-4 persisted into later stages, with 56% of ovarioles having g-H2AV labeling in both nurse cells and oocytes in stage 1, and 8% of ovarioles having g-H2AV labeling as late as stage 4 (Figure 7B, B’, Table S1). Like the p535A-1-4 (A-B-) null, females homozygous for the p53A2.3 (A-B+) allele also had more than four cells per cyst that labeled intensely for g-H2AV, as well as g-H2Av labeling in oocytes up to stage 1 in 25% of ovarioles, not as frequent as that in the p535A-1-4 (A-B-) null (56%) (Figure 7C, C’). The frequency of p53A2.3 (A-B+) ovarioles with g-H2Av labeling in stage 1 was significantly different from wild type for the oocyte but not nurse cells (see Table S1 for p values). Females homozygous for p53B41.5 (A+B-) also had more than four g-H2AV positive cyst cells and repair foci that persisted up to stage 2, later in oogenesis than in p53A2.3 (A-B+) (stage 1), but not as late as in the p535A-1-4 (A-B-) null (stage 4) (Figure 7D, D’). g-H2AV foci were not observed in egg chambers after stage 6 in any genotype, suggesting that breaks are eventually repaired in the p53 mutants. The DNA damage in the p53 mutant strains were at least as severe as those for mutants in genes required for meiotic recombination and repair, but we did not observe the egg ventralization or female sterility that have been described for those repair mutants (Ghabrial et al. 1998; Ghabrial and Schupbach 1999; Hughes et al. 2018). Altogether, these data reveal that p53A and p53B mutants have persistent DNA breaks in the Drosophila female germline.
p53A and p53B isoforms are crucial for germline genome integrity when meiotic recombination repair is compromised
To further explore the role of p53 isoforms in DNA break dynamics, we tested whether p53 plays a more prominent role in the germline when there are defects in meiotic DNA recombination. We examined females doubly mutant for p53 and okra, the fly ortholog of Rad54L, which is required for homologous recombination (HR) DNA repair in meiotic germline and somatic cells (Ghabrial et al. 1998; Sekelsky 2017; Hughes et al. 2018). It was previously reported that females doubly mutant for okra and a p53 null allele result in egg chambers with extra nurse cells and shorter eggs, which was partially suppressed by mutation of mei-W68, but the relationship of this genetic interaction to DNA break repair, and the possible role of the different p53 isoforms, have not been explored (Lu et al. 2010).
Compared to wild type, females that were transheterozygous for two mutant okra alleles (okraRU/AA) had more cells per cyst with strong g-H2AV labeling in the germarium, which abnormally persisted into germarium region 3 (stage 1), consistent with previous reports that meiotic HR repair is delayed in okra mutants (Figure 8A-B’) (Jang et al. 2003; Mehrotra and McKim 2006; Lake et al. 2013). Also consistent with previous reports, okraRU/AA females were sterile (Ghabrial et al. 1998). Analysis of the different okra;p53 double mutants indicated that they all had DNA break phenotypes that were more severe than the corresponding p53 or okra single mutants. In okraRU/AA; p535A-1-4 (A-B-) double mutants, g-H2AV foci persisted up to stage 2 in 100% of ovarioles, with some ovarioles having repair foci in nurse cells and oocyte up to stage 5-6, ~30 hour later in oogenesis than wild type (Figure 8C, C’) (Lin and Spradling 1993). The okraRU/AA ; p53A2.3 (A-B+) double mutants also had repair foci up to stage 6 in almost all ovarioles (Figure 8D, D’). Unlike okraRU/AA; p535A-1-4 (A-B-) null ovaries, however, repair foci in these okraRU/AA; p53A2.3 (A-B+) were evident in the oocyte only (8D, D’). Given that p53A2.3 expresses p53B but not p53A, these results suggest that in okra mutants the p53A isoform is required to protect genome integrity in the oocyte, while the p53B isoform is sufficient to rescue the DNA breaks in nurse cells. okraRU/AA; p53B41.5 (A+B-) also had repair foci up to stage 6, but in these ovaries lacking p53B there were numerous repair foci in both the nurse cells and oocyte (Figure 8E, E’). Thus, p53B is required in nurse cells and oocytes. These data suggest that p53 isoforms are more crucial when HR is compromised, and that they have redundant and distinct functions in nurse cells and oocytes to protect genome integrity.
p53A is required for the meiotic pachytene checkpoint
Defects in DNA recombination and persistent DNA damage are known to activate a meiotic pachytene checkpoint arrest in multiple organisms (Bahler et al. 1994; Gebel et al. 2017). During oogenesis in mice and humans, the pachytene arrest is mediated by p63 and p53 (Suh et al. 2006; Bolcun-Filas et al. 2014; Coutandin et al. 2016; Gebel et al. 2017; Marcet-Ortega et al. 2017; Rinaldi et al. 2020). In Drosophila, it is known that defects in the repair of meiotic DNA breaks activate the pachytene checkpoint, but it is not known whether this checkpoint response requires p53 (Ghabrial et al. 1998; Joyce and McKim 2011; Hughes et al. 2018). To address this question, we took advantage of a visible manifestation of the Drosophila pachytene checkpoint arrest, which is a failure of the oocyte nucleus to form a compact spherical structure known as the karyosome, which normally occurs during stage 3 of oogenesis (Ghabrial et al. 1998). We examined karyosome formation by labeling with antibodies against the synaptonemal complex (SC) protein C(3)G and DAPI (Page and Hawley 2001). In wild type females, 95% of ovarioles had a spherical compact karyosome beginning in stage 3 (Figure F, K). In okraRU/AA mutants, however, compaction was normal in only 5% of ovarioles, with the oocytes in 95% of ovarioles appearing either diffuse or ellipsoidal, morphologies that have been described in previous studies that showed that the DNA repair defects in okra mutants strongly activate the pachytene checkpoint (Figure 8 G, K) (Ghabrial et al. 1998). In okraRU/AA; p535A-1-4 (A-B-) null double mutants, however, karyosome compaction was normal in 68% of ovarioles, a fraction that is significantly different than the okra single mutant, suggesting that p53 is required for normal activation of the pachytene checkpoint (Figure 8H, K). Similarly, in okraRU/AA; p53A2.3 (A-B+) double mutants karyosome compaction was normal in 68% of ovarioles, significantly different than the fraction in okra single mutants alone (Figure 8I, K). This failure to activate the pachytene checkpoint in the majority of okraRU/AA;p53A2.3 (A-B+) ovarioles is even more striking given that they had a more severe DNA break phenotype in the oocyte than did okra single mutants that strongly engaged the checkpoint (Figure 8B, B’, D, D’). In contrast, okraRU/AA; p53B41.5 (A+, B-) mutants had normal karyosome compaction in only 9% of ovarioles, a fraction that was not significantly different than okra single mutants, indicating that the pachytene checkpoint was activated in most of these ovarioles that expressed p53A but not p53B (Figure 8J, K). Altogether these data suggest that the p53A isoform is required for normal pachytene checkpoint activation when meiotic DNA recombination is impaired, analogous to the functions of mammalian p53 and p63.
Discussion
A common property of the p53 gene family across organisms is that they encode multiple protein isoforms, but their functions are largely undefined. We found that the Drosophila p53B protein isoform is more highly expressed in the germline where it colocalizes with a shorter p53A isoform in subnuclear bodies. Despite this p53B germline expression, it is the p53A isoform that was necessary and sufficient for the apoptotic response to IR in both the germline and soma. In contrast, p53A and p53B both responded to programmed meiotic DNA breaks. Although apoptosis is repressed in meiotic cells, p53A and p53B mutants had persistent DNA breaks, a phenotype that was enhanced when meiotic DNA recombination was defective. The role of the p53 isoforms to prevent persistent DNA breaks was cell type specific, with p53B being necessary and sufficient in nurse cells, whereas both p53B and p53A were required in the oocyte. Our data has also uncovered a requirement for the Drosophila p53A isoform in the meiotic pachytene checkpoint response to unrepaired DNA breaks. Overall, these data suggest that Drosophila p53 isoforms have evolved overlapping and distinct functions to mediate different responses to different types of DNA damage in different cell types. These findings are relevant to understanding the evolution of p53 isoforms, and have revealed interesting parallels to the function of mammalian p53 family members in oocyte quality control.
p53 localizes to subnuclear bodies in Drosophila and humans
p53 isoforms colocalized to subnuclear bodies in the Drosophila male and female germline (Figure 9A). This finding is consistent with a previous study that reported p53 bodies in the Drosophila male germline, although that study did not examine individual isoforms (Monk et al. 2012). We deem it likely that these p53 bodies form by phase separation, an hypothesis that remains to be formally tested (Mitrea and Kriwacki 2016; Alberti 2017). Drosophila p53 subnuclear bodies are reminiscent of human p53 protein localization to subnuclear PML bodies (Mauri et al. 2008). Evidence suggests that trafficking of p53 protein through PML bodies mediates p53 post-translational modification and function, although the relationship between nuclear trafficking and the functions of different p53 isoforms has not been fully evaluated (Fogal et al. 2000; Chang et al. 2018). Similarly, we observed a decline in abundance of p53B within p53 bodies in germarium region 2a, followed by a restoration of p53B within bodies in region 3. This fluctuation of p53B in bodies temporally correlates with the onset of meiotic DNA breaks in region 2a and their repair in regions 2b - 3. Although p53B was undetectable in p53 bodies in many cells of region 2, the analysis of the p53B mutant alleles indicated that it is required in those cells to activate a hid-GFP reporter to wild type levels and to prevent persistent DNA breaks. These observations are consistent with the idea that nuclear trafficking of p53B out of bodies may mediate its response to meiotic breaks, although further experiments are required to test this idea (Figure 9A). Future analysis of Drosophila p53 bodies will help to define how p53 isoform trafficking mediates the response to genotoxic and other stresses.
p53A mediates the apoptotic response to IR in the soma and germline, whereas both p53A and p53B respond to meiotic DNA breaks
Our data indicate that the p53A isoform is necessary and sufficient for activation of proapoptotic gene expression and apoptosis in response to IR, in both the soma and germline. Alleles that expressed only p53A had IR-induced hid-GFP expression and apoptosis that was equivalent to wild type, whereas those that expressed only p53B were equivalent to p53 null alleles. While our manuscript was in preparation, it was reported that p53A and p53B both participate in the apoptotic response to IR in the ovary (Park et al. 2019). That study used the GAL4 / UAS system to express either p53A or p53B rescue transgenes in a p53 null background. In contrast, we created and analyzed loss-of-function, isoform-specific alleles at the endogenous p53 locus, which we believe more accurately reflect the physiological function of p53 isoforms. We favor the conclusion, therefore, that it is the p53A isoform that has the exclusive function of mediating the apoptotic response to IR in the soma and germline.
Dividing germline cystocytes in germarium region 1 apoptosed after IR, but their ancestor GSCs and descendent meiotic cells did not (Figure 9B). The expression of the hid-GFP promoter reporter in GSCs is consistent with previous evidence that apoptosis is repressed in these stem cells downstream of hid transcription by the miRNA bantam (Wylie et al. 2014; Xing et al. 2015; Ma et al. 2016). How meiotic cells repress apoptosis is not known, although it is crucial that they do so because they have programmed DNA breaks. Our finding that IR does not induce high levels of hid-GFP in meiotic cells suggests that, unlike GSCs, apoptosis is repressed in meiotic cells upstream of p53-regulated proapoptotic gene expression.
Although p53B did not mediate the apoptotic response to IR, our data indicate that both p53B and p53A respond to meiotic DNA breaks (Figure 9B). In the absence of IR, we observed p53-dependent expression of the hid-GFP reporter during early meiosis, consistent with previous evidence that p53 is activated by meiotic DNA breaks (Lu et al. 2010). While hid-GFP expression was undetectable in p53 null mutants, the p53B mutant alleles, which express p53A, reduced expression by only 50%. Together, these results suggest that both p53A and p53B respond to meiotic DNA breaks. The results for the p53A mutant allele were not informative for this hypothesis, however, because in that mutant hid-GFP expression was higher earlier in oogenesis. The mechanism for this gain of function phenotype is not clear, but a cogent hypothesis is that the p53A subunit restrains the transcription factor activity of the more potent p53B in heterotetramers, and in the p53A isoform mutants the exclusive formation of more active p53B homotetramers induces higher and precocious hid-GFP expression. This hypothesis is consistent with our previous evidence that the p53B isoform with a longer TAD is a much stronger transcription factor than p53A, and that p53A and p53B can form heterocomplexes (Zhang et al. 2015). Moreover, it is known that in humans shorter p53 isoforms repress the transcriptional activity of longer ones in heterotetramers (Anbarasan and Bourdon 2019). It is important to note, however, that while hid expression was constitutively higher in the p53A mutants than in wild type, it was lower than that induced by IR, and did not result in apoptosis. Altogether, the data indicate that p53A responds to DNA damage caused by IR, whereas both p53A and p53B respond to programmed meiotic DNA breaks. Important questions motivated by our results are how these distinct responses to DNA damage are determined by different types of DNA lesions, checkpoint signaling pathways, and p53 isoform structure.
p53 isoforms have redundant and distinct requirements to prevent germline DNA damage
p53 mutants had persistent DNA breaks in the Drosophila female germline (Figure 9B). These persistent germline DNA breaks may reflect a requirement for p53 for the timely repair of meiotic DNA breaks, and / or the prevention of new breaks by other mechanisms. Consistent with a role in meiotic DNA break repair, p53 mutants had an increased number of cells with g-H2AV foci beginning in germarium stage 2a, a time when mei-W68 induces programmed meiotic double strand DNA breaks. Moreover, g-H2AV foci were continuously observed to different later times in oogenesis depending on the p53 genotype, which was enhanced by okra (RAD54L) mutations that are known to compromise the repair of meiotic DNA breaks. These observations are consistent with the idea that meiotic DNA break repair is delayed for different amounts of time in the different p53 genotypes. It has also been shown, however, that p53 is required with the piRNA pathway to fully restrain mobile element activity in the female germline (Wylie et al. 2016; Wei et al. 2019). A non-mutually exclusive possibility, therefore, is that the increase in germline DNA breaks in the p53 mutants is also the result of elevated mobile element activity.
The proposed role for Drosophila p53 in the repair of meiotic DNA breaks is consistent with evidence from other organisms that p53 has both global and local roles in DNA repair. It is known that Drosophila p53 and specific isoforms of human p53 induce the expression of genes required for different types of DNA repair (Brodsky et al. 2004; Gong et al. 2015; Williams and Schumacher 2016). p53 also acts locally at DNA breaks in a variety of organisms, including humans, where it can mediate the choice between HR versus non-homologous end joining (NHEJ) repair (Moureau et al. 2016; Williams and Schumacher 2016). In fact, it has been shown that human p53 directly associates with RAD54 at DNA breaks to regulate HR repair, consistent with our finding that p53; okra (RAD54L) double mutants have enhanced DNA repair defects (Linke et al. 2003). Moreover, the the C. elegans p53 ortholog CED-4 locally promotes HR and inhibits NHEJ repair in the germline (Mateo et al. 2016). Thus, we deem it likely that the DNA damage that we observe in the germline of Drosophila p53 mutants may, in part, reflect a local requirement for p53 protein isoforms to regulate meiotic DNA repair (Figure 9A, B).
Our analysis also revealed that p53 isoforms have overlapping and distinct requirements to prevent persistent DNA damage in different cell types (Figure 9B). The results indicated that both p53A and p53B are required in the oocyte, whereas p53B is necessary and sufficient in nurse cells, even though nurse cells express both p53A and p53B isoforms. This differential requirement for p53 isoforms may reflect differences in how meiotic breaks are repaired in nurse cells versus oocytes. While it is not known whether DNA repair pathways differ between nurse cells and oocytes, evidence suggests that the creation of meiotic breaks does, with breaks in prooocytes but not pro-nurse cells depending on previous SC formation (Mehrotra and McKim 2006). Important remaining questions include whether different p53 isoforms participate in, and influence the choice among, different DNA repair pathways in different cell types.
Similar to the mammalian p53 family, Drosophila p53A is required for the meiotic pachytene checkpoint
Our study has also uncovered a requirement for Drosophila p53 in the meiotic pachytene checkpoint. This function was isoform specific, with p53A, but not p53B, being required for full checkpoint activation in oocytes with persistent DNA breaks. The failure to engage the pachytene checkpoint in the majority of okra;p53A2.3 double mutant oocytes is more striking given that these cells had more severe DNA repair defects than the okra single mutants that strongly engaged the checkpoint. Some okra; p53 null egg chambers had a pachytene arrest, which suggests that p53-independent mechanisms also activate the checkpoint, perhaps in response to secondary defects in chromosome structure which are known to independently trigger the pachytene checkpoint in flies and mammals (San-Segundo and Roeder 1999; Wu and Burgess 2006; Li and Schimenti 2007; Joyce and McKim 2009). Moreover, although the pachytene checkpoint was strongly compromised in the p53 null and p53A mutant alleles, they did not suppress okra female sterility, suggesting that other mechanisms ensure that eggs with excess DNA damage do not contribute to future generations. Altogether, the results indicate that p53A is required for both DNA repair and full pachytene checkpoint activation in the oocytes.
Evidence suggests that the ancient function of the p53 family was of a p63-like protein in the germline (Levine 2020). Consistent with this, our findings in Drosophila have parallels to mammals where the TAp63α isoform and p53 mediate a meiotic pachytene checkpoint arrest, and the apoptosis of millions of oocytes that have persistent defects (Di Giacomo et al. 2005; Suh et al. 2006; Bolcun-Filas et al. 2014; Gebel et al. 2017; Rinaldi et al. 2017; Rinaldi et al. 2020). Our evidence suggests that the different isoforms of the sole p53 gene in Drosophila may subsume the functions of vertebrate p53 and p63 paralogs to protect genome integrity and mediate the pachytene arrest. Unlike p53 and p63 in mammals, however, Drosophila p53 does not trigger apoptosis of defective oocytes. Instead, the activation of the pachytene checkpoint disrupts egg patterning, resulting in inviable embryos that do not contribute to future generations (Hughes et al. 2018). Thus, in both Drosophila and mammals, the p53 gene family participates in an oocyte quality control system that ensures the integrity of the transmitted genome.
Materials and Methods
Drosophila genetics
Fly strains were reared at 25 C. Fly strains were obtained from the Bloomington Drosophila Stock Centre (BDSC, Bloomington, IN, USA) unless otherwise noted. The hid-GFP fly strain was a generous gift from W. Du (Tanaka-Matakatsu et al. 2009). A y wc67C23 strain was used as wild type. The okra mutant strains were obtained from Trudi Shupbach’s lab. See Table S2 for a complete list of fly strains used in this study.
Creation and characterization of p53A and p53B isoform specific alleles
Isoform specific alleles of p53 were generated by injection of gRNA encoding plasmid into embryos of a nanos-Cas9 strain using standard methods (Gratz et al. 2013; Ren et al. 2013). Candidate lines for p53B-specific alleles were initially identified by screening for co-knockout of white (Ge et al. 2016). Injections were performed by Rainbow Transgenics (USA). Alleles were identified by PCR genotyping. The p53A2.3 allele is a 23 bp deletion (coordinates 23053346–23053368 in the D. melanogaster genome version 6.32), and has a 7 base-pair (Robin et al. 2019). The p53B41.5 allele is a 14 bp deletion (coordinates 23053726-23053739) with an insertion of a single Adenine. RT-PCR analysis of p53 isoform-specific mutants was performed on mRNA from adult flies using standard methods. See Figure S3 and Table S2 for further information about alleles, gRNAs and primers.
Gamma irradiation and cell death assays
Adult females were mated and conditioned on wet yeast for three days. They were then irradiated with a total of 4000 rad (40 Gy) from a Cesium source and were allowed to recover at 25 C for 4 hours before TUNEL labeling. TUNEL labeling (In Situ cell death detection kit, Fluorescein, Roche) was performed according to manufacturer’s instructions. Follicle cell death in Figure 2 was quantified by counting cells TUNEL positive cells in stage 6.
Immunofluorescent microscopy
Dissection, fixation, antibody labeling and immunofluorescent microscopy of testes and ovaries were as previously described (Thomer et al. 2004). Primary antibodies and concentrations used were rabbit anti-GFP (Invitrogen) 1:500, rabbit anti-dsRed (Clontech) 1:200, mouse anti dsRed (Clontech) 1:200, mouse anti Hts1B1 (DSHB) 1:20 (Zaccai and Lipshitz 1996), and mouse anti-gH2AV (DSHB) 1:1000 (Lake et al. 2013). The anti-gH2AV antibody was preabsorbed against fixed wild type ovaries before use. Secondary antibodies were Alexa 488 anti-rabbit, Alexa 488 anti-mouse, Alexa 568 anti-rabbit, and Alexa 568 anti-mouse (Jackson) all used at 1:500-1-750. Samples were counterstained with DNA dye 4’,6-diamidino-2-phenylindole (DAPI) at 1μg / ml. Confocal micrographs were captured on a Leica SP8 confocal using a 63X multi-immersion lens.
Fluorescent quantification
Hid-GFP fluorescence in Figures 3 and 4 was quantified using the LASX software of the Leica Sp8 confocal microscope. Total GFP intensity was measured across a composite z-stack of each germarium. See Figure legends for sample sizes, biological replicates, and p values.
The intensities of GFP-p53A and mCh-p53B in p53 bodies in Figures 1, 6, and S4, were quantified along a line using LASX software on the Leica SP8. For strains expressing both GFP-p53A and mCh-p53B, the ratio of GFP-p53A: mCh-p53B was quantified within each body. For those expressing a single tagged isoform, the ratios of intensities in germarium regions 1:2 or 2:3 among different bodies was calculated, all within the same germarium to control for technical variation. See figure legends for sample sizes and p values.
g-H2Av was quantified in Figures 7 and 8 by scoring cells whose total fluorescent intensity was above the threshold of wild type controls. Nurse cells and oocytes were scored as positive or negative in different stage egg chambers within each ovariole. See Figure legends and Table S1 for sample sizes and p values.
Supplemental Data
Acknowledgements
We thank W. Du, K. McKim, T. Shupbach and the Bloomington Drosophila Stock Center for fly strains; S. Hawley and J. Sekelsky for antibodies; and FlyBase for critical information. Thank you to H. Herriage, S. Brown, and M. Dykhouse for helpful discussions. Thanks to J. Powers of the IU Light Microscopy Imaging Center (LMIC) for imaging advice and support. This research was supported by NIH 2R01GM113107 to B.R.C.
Footnotes
This version corrects typographical errors discovered in the previous version.