Multiple 9-1-1 complexes promote homolog synapsis, DSB repair, and ATR signaling during mammalian meiosis

DNA damage response mechanisms have meiotic roles that ensure successful gamete formation. While completion of meiotic double-strand break (DSB) repair requires the canonical RAD9A-RAD1-HUS1 (9A-1-1) complex, mammalian meiocytes also express RAD9A and HUS1 paralogs, RAD9B and HUS1B, predicted to form alternative 9-1-1 complexes. The RAD1 subunit is shared by all predicted 9-1-1 complexes and localizes to meiotic chromosomes even in the absence of HUS1 and RAD9A. Here, we report that testis-specific disruption of RAD1 in mice resulted in impaired DSB repair, germ cell depletion, and infertility. Unlike Hus1 or Rad9a disruption, Rad1 loss in meiocytes also caused severe defects in homolog synapsis, impaired phosphorylation of ATR targets such as H2AX, CHK1, and HORMAD2, and compromised meiotic sex chromosome inactivation. Together, these results establish critical roles for both canonical and alternative 9-1-1 complexes in meiotic ATR activation and successful prophase I completion.

protein 1 (SYCP1). By pachynema DSB repair is completed and gH2AX is no longer 60 present on the fully synapsed autosomes. However, in male meiocytes, abundant gH2AX 61 is apparent at the sex body containing the X and Y chromosomes, which synapse only in 62 a small domain called the pseudoautosomal region but otherwise remain unsynapsed. 63 Meiotic cells subsequently enter diplonema, featuring dissolution of the central element 64 while homologous chromosomes remain tethered by crossovers. Breakdown of the SC or Sertoli cells. Spermatogonia also displayed relatively high levels of Atr, along with 164 Topbp1 and Etaa1 ( Figure 1C). Analysis of expression data from human testis similarly 165 showed that Hus1b and Rad9b expression was highest in spermatocytes, whereas Rad1 166 and Rad9a expression was highest in spermatogonia and Hus1 expression was highest 167 in early spermatids (Guo et al., 2018). Together these results suggest that the 9-1-168 1/TOPBP1/ATR and ETAA1/ATR signaling axes are expressed in pre-meiotic 169 spermatogonia and suggest roles for alternative 9-1-1 complexes in male meiosis. 170 To further analyze the evolutionary relationships between 9-1-1 subunits, we 171 Significant ERC values were identified between the RAD1, HUS1, and RAD9B subunits, 176 supporting the notion that alternative 9-1-1 complexes assemble in germ cells ( Figure  177 2A). These findings are consistent with reports that RAD9B physically interacts with 178 RAD1, HUS1, and HUS1B (Dufault et al., 2003), and similarly that HUS1B interacts with 179 RAD1 (Hang et al., 2002), suggesting that the paralogs contribute to alternative 9-1-1 180 complexes that include RAD9B-RAD1-HUS1 (9B-1-1) and RAD9B-HUS1-HUS1B (9B-1-181 1B) ( Figure 2B). 182 183

Testis-specific RAD1 loss leads to increased germ cell apoptosis and infertility. 184
To determine how disrupting the subunit shared by all of the 9-1-1 complexes 185 impacted meiosis, we created a Rad1 CKO model by combining a conditional Rad1 allele Immunoblotting of whole testis lysates from 12-week-old Rad1 CKO mice confirmed 195 significant reduction in RAD1 protein (n=5 control and 5 CKO; Figure 2C). The residual 196 RAD1 protein observed in Rad1 CKO mice could arise from somatic cells of the testis or 197 pre-meiotic germ cells. However, we cannot exclude the possibility that persistent RAD1 198 protein exists in spermatocytes due to partial CRE recombinase efficacy or perdurance 199 of RAD1 protein from pre-meiotic stages. Testes from Rad1 CKO males were one-third 200 the size of control testes at 4 weeks of age, while bodyweight was not altered ( Figure 2D). Next, we tested how localization of 9-1-1 subunits was affected by RAD1 loss.    leptonema and zygonema ( Figure 3D). However, 97% of pachytene-like Rad1 CKO cells 278 showed gH2AX present at asynaptic sites, with no clear presence of a sex body (n= 98 279 cells, 3 CKO mice). Interestingly, a subset of asynaptic regions in Rad1 CKO cells lacked 280 detectable gH2AX staining ( Figure 3D, white arrow heads), suggesting that the DNA 281 damage signaling at asynaptic sites was perturbed (Figure 3-figure supplement 1B). 282 Given that spermatocytes from Rad1 CKO mice exhibited significantly increased 283 asynapsis, we next assessed meiotic progression in these cells by staining for the histone 284 variant H1T and the recombination marker MLH1. First, we questioned whether Rad1 285 CKO cells were able to progress past mid-pachynema. Histone variant H1T is a marker 286 of mid-pachynema and later staged wild-type spermatocytes (Barchi et al., 2005). Control 287 cells demonstrate H1T staining as they progress into mid-pachynema (   (67 ± 44 RAD51 foci) as compared to control pachytene-stage meiocytes (10 ± 4 RAD51 327 foci) ( Figure 4F). These results for RAD51 localization in Rad1 CKO spermatocytes 328 differed from those in Hus1 CKO mice, where RAD51 appeared normal in early prophase 329 and then was aberrantly retained at a small number of sites in pachytene-stage cells 330 (Lyndaker et al., 2013a). Together these results suggest that the 9-1-1 complexes are 331 critical for proper DSB repair during mammalian meiosis and that absence of RAD1, or to 332 a lesser extent HUS1, leaves persistent unrepaired DSBs. 333 The delayed loading of MEIOB, RPA and RAD51 observed in Rad1-deficient 334 spermatocytes raised the possibility that DSB formation was impaired. To determine 335 whether the defects were related to DSB formation or the subsequent repair steps, we 336 The best characterized ATR substrate in somatic cells is the transducer kinase CHK1. 394 CHK1 has been proposed to play a role in meiotic DSB repair and is suggested to aide 395 wild-type cells, CHK1 phosphorylation (S317) occurs during leptonema and zygonema at 398 unsynapsed chromosomes. During pachynema, pCHK1 (S317) is apparent as a cloud 399 over the sex body, similar to H2AX and ATR ( Figure 5F). Interestingly, in the Rad1 CKO 400 mutant, pCHK1 was absent at all stages of prophase I. By contrast, meiotic spreads from 401 Hus1 CKO mice showed normal patterns of CHK1 (S317) and HORMAD2 (S271) 402 phosphorylation ( Figure 5E-F). That meiotic CHK1 phosphorylation is normal in the 403 absence of HUS1 but disrupted by RAD1 loss suggests that alternative 9-1-1 complexes 404 play an important role in activating the transducer kinase CHK1 during meiotic prophase 405

I. 406
The defects in ATR signaling observed in Rad1 CKO mice suggested that disruption 407 of 9-1-1 complexes might impair meiotic silencing. To test this possibility, we evaluated 408 meiotic silencing via RNA fluorescent in situ hybridization (FISH) for the X-chromosome 409 gene Scml2 that should be silenced in early pachynema-stage cells (Royo et al., 2010). 410 Scml2 expression was detected in 7.1 ± 0.6% of early pachytene control cells while Rad1 411 CKO cells showed expression in 28.9 ± 3.2% (p<0.0001; Figure 5G), indicating that 412 meiotic silencing was disrupted by RAD1 loss. The analysis of Scml2 expression focused 413 on cells with normal homolog synapsis and excluded those with asynapsis, which could underestimate the extent of the silencing defect upon RAD1 loss since some cells with 415 normal synapsis in Rad1 CKO mice retain RAD1 expression. Nevertheless, these data 416 demonstrate the importance of alternative 9-1-1 complexes in ensuring that ATR-417 mediated MSCI occurs. 418 419 Comprehensive profiling of protein phosphorylation in testes from Rad1 CKO mice. 420 Our findings that phosphorylation of key ATR substrates like HORMAD2 and CHK1 421 was disrupted in Rad1 CKO mice prompted us to more thoroughly characterize how 422 RAD1 loss impacts meiotic signal transduction. Since the 9-1-1 complex is well-423 established to regulate ATR activation, we sought to identify phosphorylation events that 424 were dependent on both the 9-1-1 complex and ATR. To accomplish this, we analyzed 425 not only Rad1 CKO testes but also those from wild-type C57BL/6 (B6) mice treated with  (1083) in wild-type spermatocytes revealed co-localization of RAD1 and pSMC3 (1083) observed on chromatin cores throughout prophase I and was phosphorylated specifically 484 at unsynapsed chromatin cores during leptonema and zygonema, and at the unsynapsed 485 regions of the XY in mid-pachynema ( Figure 7D). Although total SMC3 loading was 486 unaffected, Rad1 CKO spermatocytes showed reduced accumulation of phosphorylated 487 SMC3 (pSMC3 S1083) at unsynapsed chromatin regions in pachytene-like cells as 488 compared to mid-pachytene-stage control cells. Moreover, western blot analysis of whole 489 testis lysates confirmed that SMC3 phosphorylation (pSMC3 S1083) was significantly 490 Hus1 CKO cells had grossly normal pSMC3 (S1083) localization to the XY in pachytene-492 stage spermatocytes. Interestingly, chronic ATRi treatment caused a decrease in pSMC3 493 (S1083) localization to X and Y chromatin loops and XY cores ( Figure 7D). Similar to the 494 results in Rad1 CKO spermatocytes, pSMC3 (S1083) localization was perturbed in 495 spermatocytes from ATRi-treated mice despite the fact that SMC3 localization to 496 chromosome cores appeared normal, suggesting a specific defect in SMC3 497 phosphorylation ( Figure 7D top panel). Together these results suggest that 9-1-1 498 complexes and ATR act in conjunction to regulate meiotic cohesin phosphorylation. 499 500 Here we report that testis-specific RAD1 loss results in defective homolog 502 asynapsis, compromised DSB repair, faulty ATR signaling, and impaired meiotic 503 silencing. Previous analyses of the canonical 9A-1-1 complex in meiosis revealed that 504 loss of Hus1 or Rad9a leads to a small number of unrepaired DSBs that trigger germ cell 505 death (Lyndaker et al., 2013a;Vasileva et al., 2013). Yet, homolog synapsis, ATR 506 activation and meiotic silencing all are grossly normal in the absence of the canonical 9-507 1-1 subunits HUS1 and RAD9A. The expanded roles for RAD1 identified here are 508 consistent with its ability to additionally interact with RAD9B and HUS1B, paralogs that 509 evolved in higher organisms and are highly expressed in germ cells. Together, our results 510 support the idea that alternative 9-1-1 complexes evolved to play essential roles in meiotic 511 DSB repair, homolog synapsis, and MSCI. 512 In Rad1 CKO spermatocytes, RAD51 loading onto meiotic chromosome cores was 513 significantly reduced at leptonema and zygonema relative to controls. In control cells, 514 DSB repair is concluding and RAD51 chromatin levels are low by mid-pachynema, but 515 substantial RAD51 focus formation was still observed in pachytene-like Rad1 CKO cells, 516 suggesting major DSB repair defects. The meiotic DSB repair defects following RAD1 517 loss are similar to those previously observed in Atr loss of function mouse models. Unlike what is observed in Atr mutants and ATR inhibitor-treated mice, localization of 527 ssDNA markers MEIOB and RPA to meiotic cores was significantly reduced in the 528 absence of RAD1. The 9-1-1 complex is well-established to modulate DNA end resection, 529 having stimulatory or inhibitory effects in different contexts. In both yeast and mammals, 530 the resection-stimulatory effects of the 9-1-1 complex involve recruitment of the Exo1 and 531 Phosphorylation of some ATR targets, such as H2AX and HORMAD2, still occurred in 548 Rad1 CKO spermatocytes but only at a subset of unsynapsed chromatin regions. It 549 should be noted that HORMAD1 and HORMAD2 localized appropriately to all 550 unsynapsed regions independently of RAD1, indicating that HORMAD localization was 551 not sufficient to driving ATR signaling and highlighting essential roles for the 9-1-1 552 complexes in meiotic ATR activation, possibly through interaction with TOPBP1. Other 553 ATR substrates were more profoundly affected by RAD1 loss. CHK1 phosphorylation 554 during meiosis was absent in Rad1 CKO mice but present in Hus1 CKO mice, suggesting 555 that HUS1-independent alternative 9-1-1 complexes are necessary for meiotic CHK1 556 activation. CHK1 is required for timely ATR localization to the XY at mid-pachynema and 557 We also observed reduced SC protein phosphorylation in Rad1 CKO and ATRi-treated 563 mice, suggesting a role for the 9-1-1 complexes in mammalian SC formation. Studies in 564 S. cerevisiae show that direct interaction between the 9-1-1 complex and an SC 565 component, Red1, is required for both meiotic checkpoint signaling and SC formation 566 In addition to phosphoproteomics, we used ERC analysis to reveal potential 585 mechanistic roles for 9-1-1 subunits. ERC analysis can infer functional protein partners 586 based upon correlated rates of evolutionary change. Consistent with our 587 phosphoproteomics data, this analysis highlighted significant evolutionary correlations 588 between the genes encoding 9-1-1 complex subunits and those encoding proteins 589 involved in SC formation, such as SYCP1, SYCE1, SYCE1L and SYCE2, in addition to 590 RAD21, RAD21L and SMC1b which are involved in cohesion. Defects in homolog 591 synapsis in Rad1 CKO mice, together with the decreased cohesin phosphorylation, further implicates the 9-1-1 complexes in these key aspects of meiotic chromosome 593 structure. However, further exploration of the mechanisms underlying the interactions 594 between SC proteins, cohesin, and the 9-1-1 complexes is necessary and may provide 595 insights into the basis for the DSB repair defects in Rad1 CKO mice, as proper SC 596 formation and cohesin function is important for DSB repair (Ishiguro, 2019). 597 In mitotic cells, ATR activation is dependent on the 9-1-1/TOPBP1 axis under cellular testes were embedded in paraffin wax and sectioned at 5µm. Immunofluorescence 672 staining was used to detect LIN28 using rabbit polyclonal anti-LIN28 antibody (Abcam, 673 ab63740). Immunohistochemistry staining was used to detect TRA98 using rat 674 monoclonal anti-TRA98 antibody (BioAcademia, 73-003). TUNEL assay was performed 675 using the Apoptag ® kit (EMD Millipore) as per the manufacturer's instructions. LIN28, 676 TRA98 and TUNEL data were quantified in ImageJ by counting the number of positive 677 cells per tubule for 50 tubules of each genotype for each age group. Differences between 678 controls and Rad1 CKOs was analyzed using Welch's unpaired t-test using Graphpad. 679 680

Meiotic spreading and immunofluorescence staining 681
Meiotic spreads were prepared from 8-to 12-week-old mice as previously described 682 immunostaining, slides were blocked using 10% goat serum and 3% BSA, followed by 687 incubation overnight with primary antibody (listed in key resources table) at room 688 temperature in a humidifying chamber. Secondary antibodies were incubated at 37°C for 689 2 hours in the dark, and slides were then cover-slipped using anti-fade mounting medium 690 (2.3% DABCO, 20mM Tris pH 8.0, 8µg DAPI in 90% glycerol). Meiotic chromosomal 691 spreads were imaged with an AxioCam MRM using a Zeiss Imager Z1 microscope (Carl was performed using Welch's unpaired t-test using Graphpad Prism8.

ATR inhibitor treatment of mice 705
Wild-type B6 mice were treated via oral gavage with AZ20 (Selleck Chemicals) 706 reconstituted in 10% DMSO (Sigma), 40% Propylene glycol (Sigma), and 50% water. 707 Three different ATRi treatments were used. Chronic ATR inhibitor treatment was 708 performed by treating mice daily for 3 days with 50mg/kg AZ20 and collecting 24 hours 709 after final dose, or by treating with 2 doses of 50mg/kg AZ20 on the first 2 days and 1 710 final dose of 25mg/kg AZ20 and collecting 4 hours after the final dose . Acute ATR 711 inhibitor-treated mice were collected 4 hours after one dose of 50mg/kg AZ20.

Orthology analysis 724
Human 9-1-1 subunit sequences were used to obtain their respective orthologs from 725 Ensemble 101(2020) and/or NCBI Gene from 33 representative mammalian species. 726 Orthologs found in Ensemble having a ≥50% of both target and query sequence identity 727 and a pairwise whole genome alignment score of ≥50 were considered to have high 728 confidence. Orthologs that did not meet those criteria were considered to have low 729 confidence. Sequences only found in NCBI Gene database were considered as high 730 confidence if they were found to be syntenic. Synteny was determined based on whether 731 the gene had at least one shared neighbor gene upstream or downstream that also was 732 conserved. Species divergence across time was obtained from TimeTree website 733 (www.timetree.org). 734 735

Phylogenetic analysis 736
Protein sequences of 9-1-1 orthologs were obtained using NCBI HomoloGene. Multiple 737 alignment of protein sequences was done using Clustal Omega (1.2.2) implemented in Thornton; +I: invariable sites; +G: rate heterogeneity among sites; +F: observed amino 741 acid frequencies). Improvements were included to take account for any evolutionary 742 limitations due to conservation of protein structure and function. A nonrooted phylogenetic 743 tree was made using Maximum Likelihood interference (4 gamma distributed rate) 744     High confidence was determined if the genomic sequence had ≥50% of both target and query sequence identity, and a pairwise whole genomic alignment score of ≥50 when compared to human or if the genomic region containing the gene was syntenic with human. If an ortholog did not reach the threshold then it was annotated as low confidence (yellow). If no ortholog was found, then it was considered absent (red          ANKRD31  STAG3  RAD51B  CDC25B  ESPL1  RAD54B  SYCP1  M1AP  MSH4  DMRTC2  MLH3  HORMAD1  MEIOB  CNTD1   AURKA  BRDT  RAD21  PLK1  UBR2  SYCE2  MYBL1  RAD1  RAD9B  MOV10L1  CCNE1  TERF1  TEX19  SYCE1  BRCA2  TEX11  BRIP1  HUS1  RAD21L1  SYCE1L  HFM1   0