NAD+-dependent deacetylase SIRT1 is essential for meiotic progression and controls repair-recombination efficiency

Meiotic components and their functions have been extensively studied. Yet, the interplay between molecular factors and regulation of their functions that is brought about by post-translational modifications, specifically (de)-acetylation, is not well characterized. SIRT1, a NAD+-dependent deacetylase has been previously shown to be necessary for spermatogenesis. However, whether it has any role to play in mammalian meiosis remains to be uncovered. Our findings identify SIRT1 as a key determinant of meiotic progression. Knocking out SIRT1 specifically in meiocytes (SIRT1Δmeio) led to a delay in progression through pachytene and repair of double strand breaks. Interestingly, despite these deficits, meiotic loss of SIRT1 did not affect synapsis nor did it lead to pachytene arrest or apoptosis. Moreover, our results demonstrate that SIRT1 is required for regulating crossover frequency and its absence results in higher crossover events. Therefore, our study brings to the fore a novel regulatory factor/mechanism that is necessary for coupling of synapsis and recombination. This is noteworthy since mutations in core meiotic components result in gross defects in synapsis, repair and recombination, and very few studies have reported the differential regulation of these processes. Further, exposing SIRT1Δmeio to low/moderate doses of ©-irradiation indicated that SIRT1 might be involved in eliciting recombination checkpoint arrest and in its absence pachytene cells progress to diplotene stage, unlike in the SIRT1WT mice. Importantly, exogenous damage resulted in enhanced retention of ©H2AX in SIRT1Δmeio diplotene cells, reiterating the critical role that SIRT1 plays in regulating repair efficiency/kinetics. Molecularly, we find that SIRT1 interacts with MRN complex and lack of SIRT1 causes hyperacetylation of several non-histone proteins including the MRN components. Given that SIRT1Δmeio mice mimic MRN hypomorphs, we propose that SIRT1-dependent deacetylation of these proteins is crucial for normal meiotic progression. Taken together, our study uncovers a previously unappreciated role of SIRT1 in meiotic progression. Author Summary Meiosis is a key process in germ cell development that is essential for generating genetic diversity via recombination. It involves precise spatio-temporal orchestration of various molecular events such as chromosomal synapsis, repair and recombination. Whereas the core meiotic components are well known, upstream factors that might be important for regulating their functions and also couple the downstream processes are less explored. In this paper, we report that SIRT1, a NAD+-dependent deacetylase, is necessary for meiotic progression by identifying its role in coupling of synapsis and recombination. By generating a meiosis specific knockout of SIRT1, we show that its absence in spermatocytes leads to inefficient/delayed repair and progression through pachytene. We have also uncovered that SIRT1 exerts control over recombination (cross over) frequency. Interestingly, our findings demonstrate that SIRT1 provides protection against exogenous genotoxic stress possibly by eliciting meiotic checkpoints. Thus, this study provides both cellular and molecular insights into the importance of SIRT1 mediated protein deacetylation in governing meiosis in mammals.


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protein in orchestrating progression through meiosis [17,20]. This is particularly 93 relevant since SIRT1 expression is highest in meiotic prophase [17,20] and it has 94 been otherwise shown to be involved in DSB repair in somatic cells [25][26][27][28][29][30][31]. 95 Therefore, if/how SIRT1 affects spermatocytes at cellular and molecular levels 96 remains unknown. 97 Seminal studies have identified key components of the meiotic machinery, 98 which are essential for efficient DNA damage, repair and recombination. Mutating 99 components such as SPO11, ATM, TRIP13, DMC1 and MLH1 leads to meiotic 100 arrest, loss of meiocytes and therefore sterility [32][33][34][35][36][37][38]. Despite these, the molecular 101 basis for functional interactions between many of these factors is less understood. Nbs1 B/ B ) [39,40] or Atm -/-; Spo11 +/-; Trip13 mod/mod [41][42][43] indicated that fine-tuning 110 of activities of these proteins is critical in coupling different molecular processes such 111 as synapsis, repair and recombination. These highlight the need for further studies 112 that will not only unravel functional interactions between core meiotic components, 113 but also of efforts to identify upstream regulators. Importantly, it is intuitive to expect 114 that post-translational modification/s (PTM/s) based regulation would exert control 115 over molecular-/temporal-coupling of these processes and hence define fidelity of 116 meiosis.

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In this study, we describe the key role that SIRT1 plays in regulating meiotic 118 progression. Our findings demonstrate that loss of SIRT1 in meiocytes affects 119 efficiency of repair and recombination, without causing an arrest in spermatogenesis. 120 Notably, Sirt1 meio mice show a delay in repair and increased cross over frequencies. 121 Exposing these mice to ionizing radiation also revealed that SIRT1 is necessary to 122 elicit checkpoints in response to mild genotoxic stress. Together, this report identifies 123 SIRT1, a NAD + -dependent deacetylase, as a critical meiotic regulator that is 124 required to couple molecular processes with cellular progression through meiosis.

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Meiotic loss of SIRT1 leads to hyper-acetylation of proteins 128 Although, previous reports have alluded to a role of SIRT1 in meiosis during 129 spermatogenesis, the precise function of this NAD + -dependent deacetylase during 130 meiotic progression is still unknown. Thus, we set out to determine the phenotype of 131 meiocyte-specific loss of SIRT1 by crossing SirT1 Exon-4 lox/lox mice with Spo11-Cre 132 mice, henceforth called Sirt1 meio. (Figs 1A, 1B, S1A and S1B). To check for changes 133 in acetylation of histone and non-histone proteins, we used both site specific and pan  Chromosome spreads from Sirt1 WT and Sirt1 meio testis were stained for 151 synaptonemal complex proteins SYCP3 and SYCP1, which mark the lateral and 152 axial elements, respectively, to assess meiotic progression and synapsis. We found 153 similar number of cells in leptotene, zygotene, pachytene and diplotene stages 154 between Sirt1 WT and Sirt1 meio mice, both in young adults and during first wave of 155 spermatogenesis ( To get a quantitative measure, we binned pachytene cells into three distinct 184 categories vis-à-vis H2AX levels/pattern, as described by a previous report [46]. 185 Specifically, cells were scored as belonging to Stage-1, -2 or -3 based on whether 186 H2AX staining was cloud like, was in patches or was completely cleared from 187 autosomes, respectively ( Fig 3C). Notably, we found that Sirt1 meio germ cells were 188 represented more in Stage-1 compared to Sirt1 WT cells both at early-and mid-9 189 pachytene, and in Stage-2 at late-pachytene . Moreover,190 mice housed at AH-1 and AH-2 phenocopied each other vis-à-vis abnormal retention 191 of H2AX in Sirt1 meio mice (S3C-S3H Figs). We specifically highlight that 192 independent of the underlying mechanism; abnormal retention of H2AX clearly 193 indicated abrogated DSB homeostasis, which could have been caused by either 194 increased DSB formation or a delay in repair in Sirt1 meio mice. based on our acetylation and SIRT1-interactome results ( Fig 1E and Fig 6A), it is 213 more likely to be caused by non-histone mediators of repair. The results presented above clearly illustrated inefficient repair/recombination 230 in the absence of SIRT1 functions in spermatocytes. To gain preliminary mechanistic 231 insights, we used proteomics to map SIRT1-interactome in the testis, specifically to 232 look for factors that could explain the phenotype of Sirt1 meio mice. Interestingly, we 233 found that SIRT1 was associated with the MRN complex in the testis (Figs 6A-6C).

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Although, SIRT1 has been implicated in regulating MRN complex via NBS1 in 235 somatic cells [28,51], whether it interacts with and deacetylates other components in 11 236 the complex, and more so during meiosis, has not been investigated until now. We 237 also found MRE11 to be hyperacetylated following SIRT1 inhibition in HEK293T cells

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It is known that loss of repair/recombination factors lead to checkpoint bypass 278 during meiosis [42,43,55]. Based on the results presented above, we were tempted 279 to examine if SIRT1 was essential to induce such quality control mechanisms, 280 specifically in response to exogenous damage. We saw a dose-dependent increase 281 in the ratios of diplotene to pachytene cells in Sirt1 meio mice compared to Sirt1 WT 282 ( Fig 7C). Our results suggest that upon induced damage, unlike in the wild type, loss 283 of SIRT1 may lead to bypass of the pachytene/recombination checkpoints.

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Interestingly, compared to non-irradiated cells, irradiated diplotene cells showed 13 285 patches of H2AX and the number of these patches was significantly higher in 286 Sirt1 meio cells compared to Sirt1 WT (Fig 7D and 7E). Together, these indicated 287 persistence of damage even in diplotene and further corroborated our earlier findings 288 on the role for SIRT1 in activating and/or coupling molecular factors involved in 289 repair and recombination. Hence, we propose that in response to exogenous 290 damage, SIRT1 is required for eliciting checkpoint mechanisms, which needs to be 291 addressed in the future. In this study, we report the importance of a NAD + -dependent deacetylase 295 SIRT1 in regulating meiotic progression. Our findings reveal that SIRT1 is required to 296 couple synapsis and meiotic DSB repair/recombination, and its absence leads to 297 defective DSB repair and altered recombination frequency. Besides being one of the 298 first reports to highlight the role of SIRT1 in meiotic progression, our study posits that 299 de-/acetylation of molecular factors that govern these processes is necessary to elicit 300 checkpoints under both basal and induced DNA damage conditions.

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Loss-of-function mutants of SIRT1 in testis have indicated that it is 302 indispensable for spermatogenesis [17,[19][20][21][22]24]. Absence of SIRT1 has been 303 shown to induce apoptosis and loss of meiotic populations, for example when Sirt1 304 was knocked out using the pre-meiotic Stra8-Cre [17]. While, recent reports have 305 provided some insights into its role in post-meiotic phases [17,22], its importance in 306 meiotic progression (specifically given its high expression in spermatocytes) remains 307 to be unraveled. Our study, which has employed Spo11-Cre to knockout Sirt1 only in 308 spermatocytes, clearly shows that it plays a key role in meiotic progression. Given addressed. In this context, our study clearly illustrates that absence of SIRT1 causes 323 global hyper-acetylation of specifically non-histone proteins, and brings to the 324 forefront the need to further investigate the interplay between protein de-/acetylation 325 and meiosis in mammals. Our study also paves way for future efforts to investigate 326 the possible links between metabolic inputs and meiosis given that SIRT1 is a NAD + -327 dependent deacetylase.

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One of the key highlights of our study is loss of coupling between synapsis 329 and repair/recombination in Sirt1 meio mice (Fig 6D). Given the tight interplay 330 between synapsis and recombination, and the fact that loss of meiotic components 331 also cause synapsis defects [39,42], the current understanding of recombination-332 mediated control of progression through meiosis is limited [38,39,42,43,60].  Moreover, the pivotal role played by SIRT1 in exerting control over DSB repair was 346 evident from the phenotype of Sirt1 meio mice exposed to irradiation induced 347 exogenous damage. Sirt1 meio spermatocytes had exaggerated retention of H2AX 348 when compared to the control, symptomatic of deficient repair. Intriguingly, however, 349 at 3Gy and 6Gy of irradiation, loss of SIRT1 led to bypass of pachytene-to-diplotene 350 checkpoint, albeit with persistent damage as indicated by increased number of 351 diplotene H2AX patches in Sirt1 meio mice compared to Sirt1 WT . Therefore, our 352 results together uncover a dual role of SIRT1 in not only coupling synapsis to 353 repair/recombination, but also in activation of checkpoints following exogenously 354 induced damage (Fig. 6D).

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It was also exciting to find that meiotic loss of SIRT1 led to a significant 356 increase in crossover frequency as indicated by enhanced number of MLH1 foci.

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This is consistent with previous reports wherein increased DSBs and/or defective 16 358 repair have been associated with altered recombination frequency [49,50]. 359 Together, these are significant findings since PTM based mechanisms that elicit 360 recombination and repair checkpoints are less understood. In this regard, we 361 propose that SIRT1-dependent deacetylation might be involved in setting a threshold 362 for activation of either of these checkpoints, under both basal and exogenously 363 induced damage conditions. In the future, it will be exciting to not only investigate the 364 interplay between SIRT1 and pathways that induce these checkpoints but also in 365 general to address the relevance of de-/acetylation-mediated control of meiotic 366 progression.

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Our efforts to gain preliminary insights into possible SIRT1-dependent 368 molecular mechanisms during meiosis revealed components of the MRN complex 369 (Fig 6B). Although, SIRT1-NBS1 interaction is known [28, 51], our results clearly 370 illustrate that SIRT1 interacts with and affects acetylation of other components of 371 MRN complex as well. In this context, we would like to highlight that Sirt1 meio mice 372 phenocopy Mre11 and Nbs1 hypomorphic mutants [39], and together with the 373 molecular data, it clearly suggests that SIRT1-MRN interplay is critical for meiotic 374 progression. In the future, it will be interesting to investigate the role of de-375 /acetylation in controlling activity/localization of MRN complex during meiosis. It is 376 also likely that SIRT1 could exert control over other key players such as ATM, p53, 377 CHK2 and TRIP13 to mediate a tight coupling of synapsis, repair and recombination.

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In summary, we have discovered a novel function of SIRT1 in meiosis. Our 379 findings further highlight the importance of identifying mechanisms that affect or 380 regulate core meiotic components. Specifically, given that mutation of some of these 381 core-components lead to meiotic arrest, our results demonstrate that regulatory post- Mumbai (AH1) and IISER-Pune (AH2) were used in this study. While this was done 391 due to shifting of our mice colony, it provided us the opportunity to score for the 392 robustness of the phenotype when mice were reared in different housing conditions.

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The molecular/cellular phenotypes described in this manuscript were consistent 394 between AH1 and AH2, and results specifically obtained from either of the facilities 395 have been clearly indicated.     Sirt1 meio mice for late pachytene. Quantitations from mice at both AH-1 and AH-2.

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Students t-test used for determining statistical significance. cells. Mean ± SEM, Mann-Whitney U test done to determine statistical significance.