Sexual dimorphism in the meiotic requirement for …...2020/03/10 · 17 reported in mice, canines and a human female. Such findings raise the question of how the loss of PRDM9 is

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Sexual dimorphism in the meiotic requirement for PRDM9: a mammalian evolutionary 1

safeguard 2

Short title: 3

Sex-limited requirement of PRDM9 in mice 4

One Sentence Summary: 5

Sex-specific modulation of a meiotic DNA damage checkpoint limits the requirement for PRDM9 in 6 mammalian fertility. 7 8

Authors 9

Natalie R Powers1, Beth L Dumont1, Chihiro Emori1, Raman Akinyanju Lawal1, Catherine Brunton1, Ken 10

Paigen1, Mary Ann Handel1, Ewelina Bolcun-Filas1, Petko M Petkov1, and Tanmoy Bhattacharyya1,* 11

1. The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609, USA. 12

*Correspondence to: [email protected] 13

Abstract: 14

In many mammals, genomic sites for recombination are determined by histone methyltransferase PRMD9. Mice 15

lacking PRDM9 are infertile, but instances of fertility or semi-fertility in the absence of PRDM9 have been 16 reported in mice, canines and a human female. Such findings raise the question of how the loss of PRDM9 is 17 circumvented to maintain reproductive fitness. We show that genetic background and sex-specific modifiers can 18

obviate the requirement for PRDM9 in mice. Specifically, the meiotic DNA damage checkpoint protein CHK2 19

acts as a modifier allowing female-specific fertility in the absence of PRDM9. We also report that in the 20 absence of PRDM9, a PRDM9-independent recombination system is compatible with female meiosis and 21 fertility, suggesting sex-specific regulation of meiotic recombination, a finding with implications for speciation. 22

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Main Text: 24

Meiotic recombination generates genetic diversity and ensures the accuracy of chromosome transmission to the 25

next generation. In many organisms, recombination is not random but occurs preferentially at sites in the 26 genome known as recombination hotspots (1). In a subset of mammals, including mice and humans, the 27 positions of hotspots are determined by the specialized histone methyltransferase PRDM9—a meiosis-specific, 28 DNA-binding zinc finger protein that uniquely trimethylates both lysines 4 and 36 of histone H3 (2-6). This 29

H3K4me3/H3K36me3 double-positive signature is thought to preferentially facilitate recombination at these 30 sites, to the exclusion of other functional elements (1, 6, 7). In the absence of PRDM9, meiotic double-strand 31 breaks (DSBs) occur in normal numbers, but they localize to functional elements enriched for H3K4me3, such 32

as promoters (7). In Prdm9-deficient C57BL/6 mice (henceforth B6.Prdm9-/-), repair of these ectopic DSBs is 33 impaired, leading to prophase I meiotic arrest of both male and female germ cells, with consequent infertility 34 due to failure to produce mature gametes (8). In human males, several point mutations in PRDM9 are 35 significantly associated with non-obstructive azoospermia (9, 10). Together, these observations suggest that 36 PRDM9-dependent recombination is required for successful reproduction. 37

However, intriguing exceptions have been reported in both mice and a human female. Prdm9-deficient 38 PWD/PhJ (henceforth PWD.Prdm9-/-) male mice have normal meiotic prophase I, although they are infertile 39

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 13, 2020. . https://doi.org/10.1101/2020.03.10.985358doi: bioRxiv preprint

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due to low sperm number (PWD.Prdm9-/- females are also infertile) (11). Further, some Prdm9-/- male mice with 1

mixed genetic backgrounds are fertile, suggesting background-dependent genetic modifiers of the phenotype 2 (11). A recent report of a single fertile PRDM9-null woman shows that PRDM9 can be dispensable for fertility 3 in human females (12). Although Prdm9 is a pseudogene in the canine lineage, both male and female canids 4

reproduce successfully (13, 14). Together, these findings imply that the requirement for PRDM9 in mammalian 5 recombination is more complex than was previously imagined. These findings also suggest that a Prdm9-6 independent recombination initiation pathway must exist even in species using PRDM9; elucidating its 7 mechanisms will help decipher the genetic complexity that is likely involved. In male mice, recombination 8 hotspots on autosomes are PRDM9-dependent, but the recombination hotspots within the pseudoautosomal 9

region of the sex chromosomes are activated by a PRDM9-independent recombination initiation pathway (7). 10 This suggests that both PRDM9-dependent and -independent recombination pathways are active in tandem 11 during mammalian meiosis and might fulfill functions necessary to bypass reproductive bottlenecks due to 12 dysfunctional or absent PRDM9. Although a PRDM9-independent recombination mechanism exists in male 13 mice, it has been unknown if a similar pathway functions in females. In this study, we investigated oogenesis 14

and the effect of genetic background to determine (1) whether there is a sex-specific requirement for PRDM9-15

dependent hotspot activation, and (2) the possible mechanisms allowing organisms to circumvent the loss of 16 PRDM9 and maintain reproductive fitness. 17

Results 18

Sex-specific requirement for PRDM9-dependent histone methyltransferase activity in mice 19

We used CRISPR/Cas9 gene editing to create a point mutation in the PR/SET domain of Prdm9 (Glu365Pro, 20

henceforth Prdm9EP) in C57BL/6J (B6) mice (Fig. S1A-B). This mutation did not entirely abolish the 21 methyltransferase activity of Prdm9 in vivo but was a severe hypomorph, as evident from the ChIP-seq data for 22

H3K4me3 in homozygous mutant spermatocytes (Fig. 1A-E). There was a 7.2-fold reduction in the number of 23 detectable peaks in spermatocytes from homozygous Prdm9EP males (henceforth B6.Prdm9EP/EP) (Fig. 1B, 24 Table S1), compared to B6 spermatocytes. The intensity of detectable PRDM9-dependent peaks was also 25

sharply reduced; B6.Prdm9EP/EP showed a 6.2-fold reduction in the mean number of reads per peak relative to 26

B6 (Table S2). Indeed, these peaks were so low that they did not register in aggregation plots of H3K4me3 27 enrichment at all known PRDM9-dependent sites (Fig. 1C-E), confirming a significant alteration in PRDM9-28 dependent methyltransferase activity in B6.Prdm9EP/EP spermatocytes. 29

This mutation was not only hypomorphic, but also yielded an unanticipated phenotype: sexual dimorphism (Fig. 30 S1C). Homozygous B6.Prdm9EP/EP males exhibited the expected meiotic arrest and infertility, phenocopying 31 both the Prdm9-null condition (8) and the methyltransferase-dead Prdm9 allele recently reported (15) (Fig. 32 S1C). In contrast to wild-type B6 controls, ovaries from prepubescent B6.Prdm9EP/EP females were smaller but 33

contained all developmental stages including primordial and primary follicles (Fig.1F, S1C). In contrast to the 34 infertile males, all tested homozygous B6.Prdm9EP/EP females produced at least one—and up to five—litters 35 when mated to B6 males in fertility tests over a period of 6 months (Fig.1G-H, Tables S3 and S4). The offspring 36 were grossly normal and healthy, and those tested—males and females—were fertile. To determine whether 37

B6.Prdm9EP/EP oocytes exhibit normal cytological features of recombination, we performed 38 immunofluorescence staining on meiotic chromatin spreads from P0 ovaries, using antibodies against the 39 crossover regulator RNF212, the crossover marker MLH1, and the synaptonemal complex (SC) protein SYCP3 40

(Fig.1I). Most (~65%) of B6.Prdm9EP/EP pachytene oocytes exhibited homologous synapsis of all chromosomes 41 with numbers of crossovers similar to those in wild-type B6 control oocytes (Fig. 1I-J, Table S5). For 42 comparison to Prdm9-deficent females, we stained P0 oocytes from B6.Prdm9-/- for the crossover marker 43 MLH1 and the SC marker SYCP3. In this case, most B6.Prdm9-/- pachytene oocytes (~67%) showed 44 widespread asynapsis and a low number of MLH1 foci on synapsed chromosomes. However, about 4% of 45 B6.Prdm9-/- pachytene oocytes exhibited fully synapsed chromosomes stained with MLH1 foci (Fig.1I-J, Table 46 S5). Together, these results show that female meiosis can tolerate lower levels of active PRDM9 than male 47 meiosis, achieving sufficient recombination to ensure viable eggs. Thus, the requirement for PRDM9-dependent 48


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hotspot activation in mammalian reproduction is subject to sex-specific control. These results also provide 1

evidence that a PRDM9-independent pathway promotes crossovers in oocytes in the absence of PRDM9. 2

We next investigated whether the absence of an efficient PRDM9-dependent hotspot activation mechanism 3 promotes recombination at PRDM9-independent sites. To determine where crossover sites occur in 4

B6.Prdm9EP/EP females, we outcrossed B6.Prdm9EP mice to WSB/EiJ (WSB) mice, producing 5 B6WSBF2.Prdm9EP/EP mice. As expected, B6WSBF2.Prdm9EP/EP males were infertile with low testis weight 6 (Table S6). We backcrossed three B6WSBF2.Prdm9EP/EP females to wild-type B6 males, and genotyped both 7 the F2 females and 20 of their progeny with a genome-wide SNP array (16). This allowed us to map crossovers 8 that had occurred in the oocytes giving rise to these progeny. We excluded crossovers in the progeny that were 9

inherited from their F2 mothers, yielding 94 informative crossovers that occurred in the Prdm9EP/EP oocytes. Of 10 these, 26 occurred in intervals with no known B6 PRDM9-dependent H3K4me3 peak (27.7%). To exclude the 11 possibility that these 26 putative PRDM9-independent crossovers occurred at heretofore unrecognized PRDM9 12 binding sites on the WSB chromosomes, we searched the B6 and WSB genomic sequences in the crossover 13 regions for PRDM9 binding motifs (17). Of these 26 intervals, 17 (18.1% of total) had no novel PRDM9 14

binding motifs in the WSB sequence relative to the B6 sequence, and are considered PRDM9-independent. Of 15

the 94 total crossovers, 12 (12.8%) could be classified as clearly PRDM9-dependent based on very high-16 resolution crossover intervals containing a known hotspot (Fig. 1K). Interestingly, the B6WSBF2.Prdm9EP/EP 17

females produced more offspring than B6.Prdm9EP/EP females (avg. offspring per female per month 4.55 vs 18 2.69, respectively; Table S3), suggesting an effect of genetic modifiers from the WSB genetic background, or 19 hybrid vigor. These results provide direct evidence for PRDM9-independent recombination events occurring in 20

oocytes that gave rise to healthy offspring. 21

Genetic background and sex-limited requirement for PRDM9 in mice 22

The human case of a fertile PRDM9-null female (12), and the higher fertility in the B6WSBF2.Prdm9EP/EP 23 females, led us to examine the impact of different genetic backgrounds on meiotic recombination and fertility in 24 the face of Prdm9 deficiency. To that end, we introgressed the B6.Prdm9-null allele onto the CAST/EiJ and 25

C3H/HeJ inbred backgrounds for five generations (~5% residual heterozygosity, henceforth CAST.Prdm9-/- and 26

C3H.Prdm9-/-, respectively). These strains derive from distinct house mouse subspecies that diverged ~0.5 27 million years ago (18, 19). Strikingly, CAST.Prdm9-/- females have functional oocytes and are fertile, producing 28 grossly healthy, fertile offspring, while C3H.Prdm9-/- females, like B6.Prdm9-/- females, are infertile (Fig. 2A-29

C, Table S4). To determine the recombination status in P0 CAST.Prdm9-/- oocytes, we immunostained 30 CAST.Prdm9-/- ovarian meiotic spreads with antibodies against MLH1 and SYCP3 (Fig. 2D, Table S5). We 31 observed normal synapsis and crossover formation in ~ 96% of CAST.Prdm9-/- pachytene oocytes, although 32 there was a significant reduction in the average number of crossovers in these oocytes compared to the 33

CAST.Prdm9+/+ control oocytes (Fig 2D-E, Table S5). Interestingly, B6CASTF1.Prdm9-/- and 34 C3HCASTF1.Prdm9-/- females were fertile, while B6C3HF1.Prdm9-/- females were infertile (Fig. 2C, Table 35 S4), indicating the presence of one or more dominant modifiers in the CAST genetic background that abrogate 36 the requirement for PRDM9 in oocytes for fertility. All Prdm9-deficient males of the genetic backgrounds we 37 tested—B6.Prdm9-/- and C3H.Prdm9-/-—exhibited meiotic arrest (Fig. S2A) and infertility (data not shown). 38

However, we did observe round spermatids and elongating sperm in histological sections of some 39 CAST.Prdm9-/- testes (Fig. S2A-C), although these males were infertile. This suggests a rescue of meiotic arrest 40

in some spermatocytes, as previously observed in PWD.Prdm9-/- males (11). In conclusion, the requirement for 41 PRDM9 in recombination initiation and fertility in mice is sexually dimorphic, and is modulated by 42 background-specific genetic modifiers that lead to fertility despite PRDM9 deficiency. 43

In light of the PRDM9-independent crossovers we observed in B6WSBF2.Prdm9EP/EP females, we questioned 44 whether recombination hotspot usage is altered in the absence of PRDM9 in CAST females. To this end, we 45 crossed B6CASTF1.Prdm9-/- females with B6CASTF1.Prdm9+/- males to generate 39 B6CASTF2.Prdm9-/- 46 females (test group) (Fig. 2F). It is expected that in these F2 test progeny more than 50% of crossovers will 47 occur at unique (PRDM9-independent) crossover sites, given that female house mice typically have 48


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recombination rates higher than those of males. To resolve the PRDM9-dependent and -independent crossover 1

sites, we compared the above test progeny with a group of 36 B6CASTF2.Prdm9-/- F2 females generated by 2 crossing B6CASTF1.Prdm9+/- females with B6CASTF1.Prdm9+/- males (control group) (Fig. 2F). These F2 3 control progeny should have most or all crossovers restricted to PRDM9-dependent hotspots, due to the 4

presence of a wild-type Prdm9 allele in both parents (Fig. 2F). We observed an overlap of only ~30% (35% to 5 45% expected by random chance) between crossovers in F2 test progeny born of B6CASTF1.Prdm9-/- mothers, 6 and crossovers in F2 control progeny born of B6CASTF1.Prdm9+/- mothers (Fig. 2G). The other ~70% (378 of 7 538 crossovers) were unique and most likely PRDM9-independent crossovers contributed by the Prdm9-null 8 mothers of the test progeny (Fig. 2G). Because crossovers are mapped at low resolution in this dataset, and 9

because we do not have a detailed map of PRDM9-dependent hotspots in the CAST genome, it was not possible 10 for us to classify these crossovers as Prdm9-dependent or -independent. Overall, these data suggest a novel set 11 of crossover sites in B6CASTF1 oocytes in the absence of PRDM9. 12

To identify the CAST modifier(s) that allow Prdm9-null female fertility, we used the 75 B6CASTF2.Prdm9-/- 13 female mice to perform quantitative trait locus (QTL) mapping with the total number of ovarian follicles per 14

female as the phenotype (Fig. 2F and H, see methods for details). We chose this phenotype because the number 15

of oocytes in mature ovaries correlates with the efficacy of meiotic DSB repair and oocyte survival (20). The 16 analysis yielded a significant QTL on Chromosome 5, with a peak at ~100.4 Mb (1.5 LOD drop: 72.9-17

127.65Mb) (Fig. 2H, S2D-E). Intriguingly, this QTL contains two critical meiotic genes: ring finger protein 212 18 (Rnf212 at 108.7Mb) and checkpoint kinase 2 (Chk2 at 110.8Mb). The success of the meiotic recombination 19 process depends on the efficient repair of meiotic DSBs and crossover formation, while oocyte survival depends 20

on successful passage through a checkpoint that monitors DNA damage. The roles of RNF212 and CHK2 in 21 DNA damage surveillance in oocytes make them possible candidates that might act as genetic modifiers of 22

PRMD9 (20, 21). RNF212 is a SUMO ligase essential for crossover formation, and also mediates oocyte quality 23 control (21). It has been reported that localization of RNF212 to DSB sites acts as a “memory” of unrepaired 24 DSBs, thus promoting apoptosis of defective oocytes during the diplotene to dictyate meiotic substage transition 25

(21). Although loss of Rnf212 is compatible with survival of oocytes in spite of persistent DSBs and synapsis 26

defects, fertility is not rescued in female Rnf212 knockout mice (21). The other candidate gene, Chk2, encodes a 27 meiotic checkpoint kinase responsible for DNA damage surveillance in oocytes (20). Ablation of CHK2 28 prevents oocyte elimination in response to both radiation-induced DNA damage and persistent meiotic DSBs 29

due to genetic mutation in Trip13 (20). Both the Rnf212 and Chk2 genomic sequences are well conserved 30 between B6 and CAST mice (Fig. S3A-B); however, regulatory variants outside the genes themselves may play 31 a critical role. 32

Modulation of a meiotic DNA damage checkpoint permits female-limited fertility in Prdm9-null B6 mice 33

The first candidate modifier gene we considered was Rnf212. In meiotic spreads, RNF212 protein expression 34 and co-localization patterns were similar between CAST.Prdm9-/- meiotic oocytes and CAST.Prdm9+/+ oocytes 35 (Fig. S4). As mentioned above, crossover formation in CAST.Prdm9-/- meiotic oocytes is normal (Fig. 2D-E), 36 suggesting that the role of RNF212 in crossover formation in CAST.Prdm9-/- remains intact. While the oocyte 37 count of B6.Rnf212−/− females is normal, these mice are infertile due to an absence of crossovers (21). RNF212 38

deficiency promotes survival of oocytes with genetic and radiation-induced DNA damage, suggesting an 39 additional role for RNF212 as a pro-apoptotic cell-cycle regulator that promotes elimination of defective 40

oocytes (21). In CAST.Prdm9-/- females, delayed activation of RNF212 in this capacity could conceivably 41 promote the survival of oocytes with unrepaired DSBs, while still fulfilling its role in crossover formation. 42 Further investigation is needed to understand the exact role of RNF212 in PRDM9-dependent and -independent 43 meiotic recombination in CAST oocytes. However, because of the similarities between Prdm9+/+ and Prdm9-/- 44 CAST oocytes in both in RNF212 expression and RNF212 co-localization, we consider it an unlikely modifier-45 gene candidate. 46

To determine directly whether Chk2 deficiency allows Prdm9-null oocytes to complete meiosis, we generated 47 Prdm9-/-Chk2-/- double knockout females in the B6 genetic background (henceforth, B6.Prdm9-/-Chk2-/-). 48


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Among P0 oocytes from B6.Prdm9-/- single mutants, most exhibited widespread asynapsis and persistent 1

unrepaired DSBs, revealed by pervasive BRCA1, γH2AFX, and IHO1 signals (Fig. S5A-C). Only 4% of these 2 P0 oocytes had fully synapsed chromosomes stained with RNF212, MLH1, and MLH3 foci (markers of mature 3 crossovers) (Fig. 3A-D, S5D, Table S5). This phenotype was substantially improved in B6.Prdm9-/-Chk2-/- 4

double-mutant oocytes, with a significant increase in the number of oocytes with fully synapsed chromosomes 5 devoid of BRCA1, γH2AFX, and IHO1 (~26%, Fig. S5A-C, E). We also observed a ~threefold increase in the 6 number of dictyate oocytes in P0 B6.Prdm9-/-Chk2-/- double-mutant females, compared to B6.Prdm9-/- females 7 (Fig. S5A-D, F). Only a small number of growing follicles survived in B6.Prdm9-/- females (Fig. 3G-H). We 8 were able to isolate metaphase I oocytes from young B6.Prdm9-/- and B6.Prdm9-/-Chk2-/- females; here we noted 9

a significant increase (~25% increase) in the number of metaphase oocytes with normal chiasmata between the 10 homologous chromosomes in B6.Prdm9-/-Chk2-/- females relative to B6.Prdm9-/- females (Fig. 3E-F). We 11 conclude that CHK2 eliminated oocytes that failed to repair meiotic DSBs, but those that completed 12 recombination were not eliminated and were able to progress through folliculogenesis. Although 4% of 13 B6.Prdm9-/- oocytes at P0 had a full complement of crossovers, this is probably not enough for fertility, 14

especially given further oocyte elimination after P0 during follicle formation and atresia. We propose that the 15

removal of Chk2 allowed more Prdm9-/- oocytes to escape the meiotic DNA damage checkpoint and 16 subsequently complete DSB repair, resulting in a higher oocyte reserve. 17

To test the impact of improved meiotic phenotypes on oocyte number and fertility, we estimated the oocyte 18 number in B6.Prdm9-/-Chk2-/- females and performed fertility tests with wild-type B6 males. In contrast to 19 B6.Prdm9-/- females, B6.Prdm9-/-Chk2-/- females produced at least one litter of grossly healthy, fertile offspring 20

over a six-month period (Fig. 3I-J, S6A, Table S4), indicating some functional ovarian reserve. However, there 21 was a high incidence of pregnancy losses and perinatal deaths (Fig. S6B), suggesting that oocyte quality is 22

compromised, consistent with the observations of metaphase oocytes in B6.Prdm9-/-Chk2-/- females. The extent 23 of this fertility was much lower than that observed in CAST.Prdm9-/- females. In contrast to females, Chk2 24 deficiency did not rescue fertility in B6.Prdm9-/- males; both single- and double-mutant males were infertile 25

with complete meiotic arrest (Fig. S5G). Together, these results demonstrate that, in females, a Prdm9-26

independent recombination pathway can give rise to the required number of crossovers for production of 27 healthy offspring, facilitated by inactivation of the Chk2-dependent DNA damage surveillance mechanism. 28

Discussion 29

In this study, we find that female meiosis tolerates critically low levels of catalytically active PRDM9, or even 30 no PRDM9 at all in some genetic backgrounds, because a PRDM9-independent recombination pathway can 31 compensate successfully. The meiotic DNA-damage repair protein CHK2 acts as a modifier, with its 32 modification or abrogation allowing completion of meiosis and fertility in females lacking active PRDM9. This 33

is not the case for males; Prdm9EP/EP and Prdm9-null males in all genetic backgrounds examined were infertile. 34 Overall, these results demonstrate that the requirement for functional PRDM9 during meiosis varies 35 substantially by genetic background as well as by sex, and that there are alternative pathways for effective 36 recombination. 37

As shown by our genetic analyses, modification or even abrogation of the CHK2-dependent DNA damage 38 checkpoint activation is a critical determinant of female fertility in the absence of PRDM9. CHK2 triggers 39 elimination of oocytes with persistent DNA damage. It is possible that PRDM9-independent DSB repair is 40

inefficient or prolonged, which would cause DSBs to persist past the timely activation of the CHK2-dependent 41 DNA damage checkpoint, leading to oocyte elimination. Ablation of Chk2 and consequent elimination of 42 checkpoint surveillance may promote survival of oocytes by allowing extra time for PRDM9-independent 43 crossovers to accumulate, permitting a sufficient number of oocytes to complete meiosis. However, this is not a 44 fully adequate explanation of the modifier effect, because although B6.Prdm9-/-Chk2-/- females were fertile, they 45 were not as productive as CAST.Prdm9-/- females. Thus, while CHK2 is clearly mechanistically involved, it 46 does not completely explain the disparity between B6 and CAST female fertility in the absence of PRDM9. 47 Furthermore, the predicted causative CAST variant(s) in Chk2 seem to be dominant and possibly neomorphic, 48


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unlike the recessive null mutation that restores fertility in B6.Prdm9-/-Chk2-/- females. It is therefore possible 1

that both Chk2 and Rnf212 are modifiers of the PRDM9-independent fertility phenotype, or that additional 2 CAST genome variants affect timing or efficacy of DNA repair, the checkpoint, or other features of meiosis that 3 determine whether or not oocytes activate the checkpoint. It may also be the case that repair of PRDM9-4

independent DSBs is more efficient in the CAST genetic background than in the B6, with fewer oocytes 5 exhibiting a number of DSBs sufficient to activate DNA damage checkpoint. Ability to repair DSBs may be a 6 limiting factor in B6 mice; the high pre- and perinatal death rate (Fig. S6B) and the aneuploidy rate we observed 7 in developing oocytes (Fig. 3E and F) indicate that, in B6 mice, the PRDM9-independent pathway leads to 8 maturation of defective oocytes. Although similar modification of an infertility phenotype via removal of Chk2 9

was reported for Trip13-deficient females (20), there are critical differences between causes of infertility in 10 Trip13- and Prdm9-deficient females. In Trip13 mutants, crossovers originate from PRDM9-dependent DSBs, 11 and complete synapsis is achieved (20, 22); in contrast, Prdm9-deficient oocytes exhibit widespread 12 chromosomal asynapsis, and form crossovers at ectopic DSBs. Trip13-deficient oocytes are eliminated due to 13 inefficient repair of DSBs in a non-crossover pathway (20). Removal of Chk2 in Trip13 mutants reestablishes 14

ovarian reserve and fertility by allowing oocyte survival and additional time for DSB repair (20); however in 15

Prdm9-deficient oocytes, Chk2 removal must also allow chromosomal synapsis and recombination at ectopic, 16 non-PRDM9 DSBs. To our knowledge, this is the first evidence that modulation of a DNA damage checkpoint 17

protein can allow survival of oocytes that undergo genetic recombination via a PRDM9-independent pathway. 18

While checkpoint modulation appears to be a feature allowing oocyte, but not spermatocyte, survival in the 19 absence of PRDM9, at least in certain genetic backgrounds, the question of the genetic and cellular mechanisms 20

behind this dimorphism is undoubtedly more complex. Although recombination is essentially the same process 21 in both sexes, spermatogenesis and oogenesis are fundamentally different cell differentiation programs. Two 22

key differences unique to males that might explain the sexually dimorphic requirement for PRDM9 are meiotic 23 sex chromosome inactivation (MSCI), which is the silencing of most genes on the sex chromosomes from 24 pachynema of meiotic prophase I into spermiogenesis (23), and deployment of Piwi-interacting RNAs 25

(piRNAs) (24, 25). MSCI is triggered by XY asynapsis in spermatocytes and is marked by sequestration of the 26

sex chromosomes in the ‘sex body’ (23). Spermatocytes undergoing PRDM9-deficient meiotic arrest in B6 27 mice do not form a normal sex body, as unsynapsed autosomes and unrepaired DSBs compromise recruitment 28 of silencing factors to sex chromosomes (8, 26, 27). This could potentially lead to failure of MSCI, triggering 29

meiotic arrest of PRDM9-deficient spermatocytes (8, 26). Inappropriate critical gene silencing on the asynapsed 30 autosomes due to meiotic silencing of unsynapsed chromatin (MSUC) may also contribute to spermatocyte 31 elimination (23). Oocytes, which are affected by MSUC to a lesser extent, may escape these outcomes (23). In 32 mammals, piRNAs are necessary to suppress expression of L1 retrotransposons during spermatogenesis (24, 33

25). Mutation or deficiency of the piRNA pathway contributes to male-limited sterility (28-30), and it was 34 recently reported that the expression of piRNAs is misregulated in Prdm9-null spermatocytes (31). Thus, it is 35 possible that dysregulation of the piRNA pathway contributes to the post-meiotic problems in Prdm9-null germ 36 cells that undergo partial meiotic arrest, as seen in PWD (11), CAST, and other genetic backgrounds (this 37 report). 38

In addition to invoking intriguing meiotic mechanisms, the sexually dimorphic responses to PRDM9 deficiency 39 described here have significant evolutionary implications. Prdm9 is the first and only known mammalian 40

speciation gene (11). Prdm9 interacts with an unknown element on the X chromosome to cause male-limited 41 meiotic arrest and hybrid sterility in F1 hybrids between the females of the M. m. musculus strain PWD/PhJ, 42 and the males of certain M. m. domesticus strains, including B6 (32, 33). Sterile (PWDXB6)F1 hybrid males 43 exhibit a significant enrichment of DSBs at PRDM9-independent hotspots and high rates of autosomal 44 asynapsis that trigger pachytene checkpoint activation (33, 34). Chromosome asynapsis is also observed in F1 45 hybrid females, although the phenotype is markedly less severe than in F1 hybrid males, and females remain 46 fertile (33, 35). Our findings suggest that F1 hybrid females may retain fertility by effectively utilizing a 47 PRDM9-independent DSB repair mechanism to evade checkpoint activation. Our work further nominates Chk2 48


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as a key modifier of these sex and strain differences in the meiotic tolerance for PRDM9-independent DSB 1

repair. 2

The pattern of male-limited hybrid sterility in the (PWDxB6)F1 model follows Haldane's rule, which postulates 3 that when only one sex of an interspecies hybrid experiences sterility or inviability, it is the heterogametic sex 4

(36, 37). Many hypotheses, largely based on role of sex chromosomes, have attempted to explain Haldane's 5 rule, including the faster-male theory, the faster X theory, meiotic drive, and the dominance theory (38). 6 However, the mechanistic basis for sexually dimorphic hybrid sterility remains unclear in most documented 7 cases. The sexually dimorphic requirement for PRDM9 for fertility and the relaxed stringency of DNA damage 8 surveillance in females that we report here provide a possible explanation for Haldane's rule in the 9

(PWDXB6)F1 hybrid sterility model. Notably, although hybrid sterility in this system depends on a genetic 10 interaction between Prdm9 and an X-linked locus (33, 35), the sex-specific manifestation of this sterility 11 appears to be driven, at least in part, by sex and genetic differences at an autosomal gene, Chk2. 12

Finally, Prdm9 is a fundamental evolutionary innovation that is thought to have evolved to direct recombination 13

away from functional elements, safeguarding these regions from recombination-associated mutagenesis (7). As 14 in all cases of evolutionary innovation, however, there are trade-offs. Use of a DNA-binding protein to specify 15

regions of recombination requires that sufficient numbers of the cognate binding sites of that protein remain 16 intact. However, PRDM9-dependent hotspots can extinguish themselves via gene conversion, leading to gradual 17

erosion of the hotspot-binding motifs recognized by a particular PRDM9 variant and, ultimately, meiotic failure 18 and infertility (39, 40). One mechanism through which populations may avoid this fate is the emergence of new 19 Prdm9 alleles that recognize new suites of hotspots (39, 40). Indeed, Prdm9 is highly polymorphic and its 20

DNA-binding domain is rapidly evolving via positive selection (1). Our findings introduce an additional, novel 21 complexity to the dynamic interplay between hotspot erosion and reproductive fitness; i.e., sex differences in 22

the usage or effectiveness of PRDM9-independent pathways may render one sex more resilient to the loss of 23 PRDM9 activity, leading to potential sex-specific fitness effects of hotspot erosion. Further work is required to 24 elucidate the mechanistic details of the sexually dimorphic responses to lack of PRDM9; nevertheless, the 25

theoretical implications raised by this phenomenon present fascinating new insights into the checks and 26

balances that constrain one of the most fundamental evolutionary processes in mammals—genetic 27 recombination. 28

References and Notes: 29

1. K. Paigen, P. M. Petkov, PRDM9 and Its Role in Genetic Recombination. Trends Genet 34, 291-300 30 (2018). 31

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Acknowledgments: We thank all members of the Bolcun-Filas, Handel, Paigen and Petkov labs for insightful 32 discussions and suggestions. We acknowledge the expertise and contributions of the Jackson Laboratory 33

Scientific Services, including the Histology core, the Microscopy core, Genome Technologies, Genetic 34 Engineering Technology and Mouse Resources for their expertise and help during this project. We thank 35 the Knockout mouse project (KOMP2) at the Jackson Laboratory for providing mice. We thank Drs. 36 Attila Toth, Paula Cohen and Neil Hunter for sharing their antibody resources; Funding: T.B. was 37 supported in part by a JAX Scholar award (19042802-15-3); N.R.P were was supported in part by 38

NICHD T32 Training Program in Developmental Genetics (T32 HD007065 to the Jackson Laboratory). 39

This work was also supported by grants from the NIH: P01 GM99640 to M.A.H and K.P, R01 40

HD093778 to E.B-F, R01 GM125736 to P.M.P and P30 CA034196 to the Jackson Laboratory for 41 scientific services.; Author contributions: Conceptualization, T.B. and N.R.P; Methodology, T.B., 42 N.R.P., B.L.D and E.B-F.; Investigation, T.B., N.R.P.,B.L.D; C.E, R.A.L., and C.B.; Writing – Original 43 Draft, T.B and N.R.P.; Writing – Review & Editing, T.B., N.R.P.,B.L.D; C.E, R.A.L., C.B; K.P; E.B-F; 44 P.M.P and. M.A.H; Funding Acquisition, T.B., N.R.P.,B.L.D;. K.P; E.B-F; P.M.P and. M.A.H; 45 Resources, T.B, N.R.P, B.L.D; K.P; E.B-F; P.M.P and M.A.H.; Supervision, T.B, B.L.D; E.B-F; P.M.P 46 and M.A.H. Competing interests: The authors declare no competing interests. Data and materials 47 availability: All data is available in the main text or the supplementary materials. All data, code, and 48

materials used in the analysis are available in some form to any researcher or commercially for purposes 49


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of reproducing or extending the analysis. H3K4me3 ChIP seq data are available at NCBI Gene 1

Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo) under accession number GSE144144. 2

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Supplementary Materials: 5

Materials and Methods 6

Figures S1-S6 7

Tables S1-S6 8

References (41-48) 9

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Fig. 1. Prdm9EP/EP females in the C57BL/6J genetic background are fertile. (A) Genome browser snapshot of 1

H3K4me3 ChIP-seq peaks in wild-type and Prdm9EP/EP spermatocytes. One strong and one weaker PRDM9-2 dependent hotspot are shown, together with one PRDM9-independent peak. (B) Venn diagram showing the 3 number of recombination hotspots with a detectable H3K4me3 peak in wild-type (B6) and Prdm9EP/EP 4

spermatocytes. (C) MA plot comparing Prdm9EP/EP vs. wild-type signal at PRDM9-dependent H3K4me3 peaks 5 that were detectable in Prdm9EP/EP spermatocytes (n = 2,591). (D) Aggregation plot showing normalized 6 average signal intensity (reads per million, RPM) at known PRDM9-dependent H3K4me3 ChIP-seq peaks (n = 7 18,838) in wild-type and Prdm9EP/EP spermatocytes. (E) Aggregation plot showing normalized average signal 8 intensity (reads per million, RPM) at non-hotspot H3K4me3 ChIP-seq peaks (n = 56,030) in wild-type, and 9

Prdm9EP/EP spermatocytes. (F) PAS-stained sections from 3-week postpartum ovaries in wild-type and 10 Prdm9EP/EP mice in the B6 genetic background. Arrows show primary follicles. Bar = 100 μm. (G) Pups 11 produced by Prdm9EP/EP female mice. (H) Litter sizes in wild-type, Prdm9-/- and Prdm9EP/EP female mice. (I) 12 Co-immunolabeling detection of RNF212 foci (red, top row), MLH1 (red, bottom row) and SYCP3 (blue) in 13 pachytene oocyte chromatin spreads in wild-type and mutant females. White arrowheads highlight unsynapsed 14

regions of chromosomes. Scale bars represent 10 µm. (J) Violin plot with dots showing numbers of MLH1 foci 15

per meiotic oocyte (error bars, SEM), in wild-type, Prdm9-/-, and Prdm9EP/EP mice. P-values were calculated 16 using Mann-Whitney U test with Tukey’s multiple testing corrections. (K) Diagram showing proportions of 17

PRDM9-dependent and -independent crossovers in progeny of B6WSBF2.Prdm9EP/EP females (n = 94). 18

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Fig. 2: CAST/EiJ Prdm9-null females are fertile. (A) Immunofluorescence staining of histology sections from 2

3-week postpartum ovaries in mice of different genetic backgrounds. DDX4 (red, also known as MVH) marks 3

oocytes. DNA is stained with DAPI (blue). Arrowheads indicate primordial follicles. Bar = 100 μm. Oocyte 4

depletion in Prdm9-/- mice in the B6 and C3H genetic backgrounds is seen in contrast to survival of oocytes in 5

wild-type and Prdm9-/- mice in the CAST genetic background. (B) Pups produced by CAST.Prdm9-/- female 6

mice. (C) Reproductive productivity of wild-type and Prdm9-/- females in different genetic backgrounds. (D) 7

Co-immunolabeling detection of MLH1 foci (red) in pachytene oocyte chromatin spreads, also labeled with 8

antibody against SYCP3 (blue) in wild-type and Prdm9-/- CAST females. Scale bars = 10 µm. (E) Violin plot 9

with dots showing numbers of MLH1 foci per oocyte (error bars, SEM). The genotypes of mice tested are 10

indicated below the graphs. P-values were calculated using Mann-Whitney U test with Tukey’s multiple testing 11

corrections. (F) Scheme of construction of B6CASTF2.Prdm9-/- female cohorts used for SNP array genotyping 12

(GigaMUGA). Seventy-five F2 females were generated by crossing B6CASTF1.Prdm9-/- and 13

B6CASTF1.Prdm9+/- females with B6CASTF1.Prdm9+/- males. Eight-week-old B6CASTF2.Prdm9-/- females 14

were phenotyped for oocyte quantity and genotyped. (G) Venn diagram showing common crossover sites 15

between F2 females generated by crossing B6CASTF1.Prdm9-/- (test, blue) and B6CASTF1.Prdm9+/- (control, 16

green) females with B6CASTF1.Prdm9+/- males. 160 common crossover sites are highlighted by overlap. Total 17

of observed crossover sites are highlighted in the center of each circle. N represents total number of F2 females 18

analyzed in each group. (H) Quantitative trait locus (QTL) mapping, with the total number of ovarian follicles 19

(refer to methods for details) per B6CASTF2.Prdm9-/- female as the phenotype. QTL (pink) reached 20

significance on Chromosome 5, with a peak at ~100.4 Mb (1.5 LOD drop: 72.9-127.65Mb, mm10). 21

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Fig. 3: Chk2 ablation rescues fertility in Prdm9-null females in the B6 genetic background. (A-D) Co-1

immunolabeling detection of MLH3 foci (red, A), MLH1 (red, C) and SYCP3 (blue, both A and C) in 2

pachytene oocyte chromatin spreads from wild-type and mutant females. Yellow arrowheads highlight 3

unsynapsed regions of chromosomes. Scale bars = 10 µm. Violin plot with dots showing numbers of MLH3 (B) 4

and MLH1 (D) foci per meiotic oocyte (error bars, SEM). The genotypes of mice tested are indicated below the 5

graphs. p values were calculated using Mann-Whitney U test with Tukey’s multiple testing corrections. (E-F) 6

Chromosome configuration in meiosis I metaphase analyzed by chromosome spreads from different mutant and 7

control oocytes (E) and quantification (F). In E, DNA (blue) and kinetochores (red) were detected by DAPI and 8

CREST antiserum, respectively. Scale bars = 10 μm. Multiple univalents in a B6.Prdm9-/- oocyte are indicated 9

with white arrowheads. The examples of oocytes from B6 and B6.Prdm9-/- Chk2-/- mice reveal all chromosomes 10

organized in bivalents. (G) PAS-stained histological sections of 3-week postpartum ovaries from mice of 11

different genetic backgrounds. Bar = 200 μm. (H) Oocyte quantification in mutant and control animals. (I) Pups 12

produced by B6.Prdm9-/- Chk2-/- female mice. (J) Litter sizes in mutant and control females. 13

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Sexual dimorphism in the meiotic requirement for …...2020/03/10 · 17 reported in mice, canines and a human female. Such findings raise the question of how the loss of PRDM9 is