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1 Title: Mammalian SWI/SNF chromatin remodeler is essential for reductional meiosis in males 2 3 Debashish U. Menon & Terry Magnuson 4 5 Department of Genetics, and Lineberger Comprehensive Cancer Center, The University of North 6 Carolina at Chapel Hill, Chapel Hill, North Carolina, NC 27599-7264, USA 7 *Correspondence: [email protected], Tel 919-962-1319, Fax 919-843-4682 8 9 Abstract: 10 BRG1, a catalytic subunit of the mammalian SWI/SNF nucleosome remodeler is essential for 11 male meiosis1. In addition to BRG1, multiple subunits (~10-14) some of which are mutually 12 exclusive, constitute biochemically distinct SWI/SNF subcomplexes, whose functions in 13 gametogenesis remain unknown. Here, we identify a role for the PBAF (Polybromo - Brg1 14 Associated Factor) complex in the regulation of meiotic cell division. The germ cell-specific 15 depletion of PBAF specific subunit, ARID2 resulted in a metaphase-I arrest. Arid2cKO metaphase- 16 I spermatocytes displayed defects in organization and spindle assembly. 17 Additionally, mutant centromeres were devoid of Polo-like kinase1 (), a known regulator of 18 the spindle assembly checkpoint (SAC)2. The loss of PLK1 coincided with an abnormal 19 chromosome-wide expansion of centromeric chromatin modifications such as 20 threonine3 phosphorylation (H3T3P) and Histone H2A threonine120 phosphorylation 21 (H2AT120P) that are critical for chromosome segregation3,4. Consistent with the known role of 22 these histone modifications in chromosome passenger complex (CPC) recruitment, Arid2cKO 23 metaphase-I display defects in CPC association. We propose that ARID2 facilitates 24 metaphase-I exit by regulating spindle assembly and centromeric chromatin. 25 26 Keywords: PBAF remodeler, ARID2, male reductional meiosis, spindle assembly, H3T3P, 27 H2AT120P 28

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29 Main text: 30 We previously demonstrated a role for the mammalian SWI/SNF chromatin remodeling complex 31 in meiotic progression by conditionally ablating its ATPase subunit, BRG1, in the testis1,5. Given 32 that SWI/SNF complexes are biochemically heterogenous6,7, it is possible that certain 33 subcomplexes might selectively govern distinct spermatogenic processes. To address this 34 possibility, we examined existing RNA-seq data generated from purified spermatogenic cell 35 populations8 and monitored the temporal expression profiles of SWI/SNF subunits that define two 36 subcomplexes, namely, BAF (Brg1 Associated Factor) and PBAF (Polybromo-BAF) (Fig. 1a). Of 37 particular interest were the ARID (AT-rich interaction domain) containing SWI/SNF subunits, 38 known to bind DNA with loose specificity9. Among the subunits examined, the mRNA expression 39 of PBAF DNA binding subunit10, Arid2, drew our attention as it peaked at pachynema (Fig. 1a). 40 More recent single cell RNA seq data also identified Arid2 as an early pachytene marker11, 41 suggesting a role in meiosis-I. We therefore performed immunostaining on testes cryosections 42 to profile ARID2 abundance in prophase-I spermatocytes staged by γH2Ax. Consistent with its 43 mRNA profile, ARID2 went from being undetectable to expressed weakly, during the transition 44 from pre-leptonema to zygonema, but appeared elevated at pachynema which is distinguished 45 by the sex body12. Additionally, ARID2 was also detected in round spermatids, enriched proximally 46 to chromocenters (Fig. 1b). To investigate its role in meiosis we conditionally knocked out Arid2 47 using the spermatogonia specific Stra8-Cre transgene13, resulting in significant depletion of 48 ARID2 and smaller testes in Arid2cKO relative to Arid2WT (Extended Data Fig. 1a , Fig. 1c). A 49 histological examination revealed a striking loss of haploid spermiogenic cell populations and 50 absence of mature spermatozoa in Arid2cKO relative to Arid2WT adult seminiferous tubules and 51 cauda epididymides respectively (Fig. 1d, Extended Data Fig .1b). The Arid2cKO testis were 52 characterized by an accumulation of stage XII tubules which is indicative of a meiotic arrest (Fig. 53 1d). While meiotic prophase-I appeared unhindered, we noticed a striking accumulation of 54 metaphase - I spermatocytes in Arid2cKO testis (Fig. 1d, e, Extended Data Fig .1c). The near 55 absence of secondary spermatocytes and spermiogenic cells in Arid2cKO testis, indicate that 56 ARID2 is essential for meiotic cell division. 57 58 Consistent with a putative role in cell division, we were able to detect abundant expression of both 59 ARID2 and BRG1 in metaphase-I spermatocytes by immunofluorescence (IF). They appeared 60 localized peripherally to the metaphase plate displaying limited chromosomal association 61 (Extended Data Fig .1d, top row). While transitioning to -I, both ARID2 and BRG1 were

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62 detected at the spindle mid-zone while maintaining their peripheral association with segregating 63 chromosomes (Extended Data Fig .1d, bottom row). The primarily cytosolic distribution of 64 SWI/SNF combined with its narrow localization to chromosomes might suggest a preferential 65 association with the spindle and or . Based on the mutant phenotype and spatial 66 distribution of PBAF subunits in late meiosis-I spermatocytes, we hypothesized that ARID2 67 regulates processes essential for . These include chromosome 68 condensation, alignment and - attachment. 69 We began by monitoring phosphorylated histone H3 on serine 10 (H3S10P), a known marker of 70 chromosome condensation and b-Tubulin which constitutes the , in Arid2WT and 71 Arid2cKO seminiferous tubules. While H3S10P levels between Arid2WT and Arid2cKO metaphase-I 72 spermatocytes looked similar, certain mutant metaphase-I chromosomes appeared rounded and 73 failed to congress at the metaphase plate (Fig. 2a, 3rd row). These defects prompted us to 74 examine meiotic metaphase spreads where we observed no signs of pairing abnormalities or 75 in Arid2cKO spermatocytes (Extended Fig .2a). More strikingly, 52 % of the scored 76 metaphase-I spermatocytes from Arid2cKO tubules lacked a normal spindle (Fig. 2a, 3rd row). 77 Metaphase-I spermatocytes that lacked spindle were demonstrably deficient in ARID2 relative to 78 those that displayed spindle in Arid2cKO tubules (Extended Fig. 2b). The latter therefore represent 79 cells that might have undergone inefficient knockout and were considered internal controls. 80 Therefore, Arid2cKO metaphase-I spermatocytes feature aberrant spindle assembly and abnormal 81 chromosome organization. 82 83 Normally, a failure to establish microtubule – kinetochore attachments would activate the spindle 84 assembly checkpoint (SAC) which delays metaphase exit 14,15. Given that Arid2cKO spermatocytes 85 are deficient in we were curious to determine the status of SAC proteins many of 86 which associate with kinetochores. For this purpose, we chose Polo-like kinase1 (PLK1), a well- 87 known SAC regulator2,16 that also influences spindle assembly17–22. In control (Arid2Het) 88 metaphase-I spermatocytes, PLK1 foci were seen overlapping with Centromere protein A 89 (CENPA), a known histone H3 variant and component of functional centromeres23 (Fig. 2b). In 90 contrast, 60 % of the scored metaphase-I spermatocytes in Arid2cKO tubules lacked centromeric 91 PLK1 (PLK1-, Fig. 2b). Those that still displayed centromeric PLK1 (PLK1+) appeared to express 92 ARID2 at almost normal levels, indicating that they were internal controls (Extended Fig. 2c). 93 Since PLK1 is known to be expressed prior to metaphase-I24, we monitored PLK1 in diplotene 94 spermatocytes where desynapsing axial elements are marked by HORMAD125. Here, PLK1 foci

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95 appeared stable in ARID2 deficient cells (Extended Fig. 2d). Therefore, the destabilization of 96 PLK1 was specific to Arid2cKO metaphase-I spermatocytes. Interestingly, PLK1 was detected in 97 ARID2 and BRG1 co-immunoprecipitations (Co-IP) with nuclear lysates from spermatogenic cells 98 harvested at 3-weeks post-partum (Fig .2c). This suggests that PBAF might facilitate the 99 recruitment of PLK1 to centromeres. 100 101 Given that PLK1 is known to influence centromeric chromatin during 26, we were curious 102 to determine the impact of ARID2 on histone modifications known to regulate cell division. These 103 include Histone H3 threonine3 phosphorylation (H3T3P) and Histone H2A threonine120 104 phosphorylation (H2AT120P), that are concomitantly deposited by HASPIN kinase3,27 and spindle 105 checkpoint kinase BUB14 respectively. Interestingly, perturbation of H3T3P levels have been 106 shown to be detrimental to oocyte maturation in mouse 27. Therefore, we first monitored H3T3P 107 in Arid2WT (normal) metaphase-I spermatocyte squashes. Similar to metaphase-I oocytes, H3T3P 108 overlapped centromeres and was distributed along the inter- chromatid axes (ICA) (Fig. 3a, 1st 109 row). In Arid2cKO preparations, the absence of centromeric PLK1 was used to differentiate the 110 mutant metaphase-I spermatocytes from internal controls (Fig .2b, Extended Fig. 2c). H3T3P 111 distribution was limited in internal controls (10%) resembling normal metaphase-I spermatocytes 112 (Fig. 3a, 2nd row). In contrast to normal spermatocytes and internal controls, H3T3P was spread 113 chromosome-wide in majority of mutant metaphase-I spermatocytes (60%), with fewer mutants 114 (30%) displaying low levels of chromatid bound H3T3P (Fig. 3a, row 3,4). We also monitored 115 H3T3P abundance in spermatogenic histone extracts obtained from control and Arid2cKO testes 116 at time points spanning the appearance of metaphase spermatocytes. By western blot, we failed 117 to notice enhanced H3T3P levels upon the loss of ARID2 (Extended Fig. 3a, top panel). Therefore, 118 the chromosome-wide expansion of H3T3P in mutants is unlikely to occur from increased HASPIN 119 activity. 120 Next, we determined the impact of ARID2 on H2AT120P. During mitosis, H2AT120P is restricted 121 to centromeres4,28. A similar centromeric enrichment of H2A120P was seen in Arid2Het (control) 122 metaphase-I spermatocytes (Fig. 3a, b, 1st row). In contrast, mutant metaphase-I spermatocytes 123 (62%) exhibited ectopic spreading of H2AT120P (Fig. 3b, 2nd row). The loss of ARID2 did not alter 124 total H2AT120P abundance, indicating normal BUB1 activity in the mutant (Extended Fig. 3b). 125 Therefore, ARID2 is required to ensure the centromeric enrichment of both H3T3P and 126 H2AT120P.

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127 In addition to centromeric chromatin we also determined whether flanking pericentric chromatin, 128 normally marked by H3K9me329 was impacted in the absence of ARID2. Like the centromeric 129 marks, total H3K9me3 abundance was not influenced by the loss of ARID2 (Extended Fig. 3a, 130 bottom panel). However, many mutant metaphase-I spermatocytes from Arid2cKO testis displayed 131 H3K9me3 spreading (38%) with the remaining exhibiting normal pericentric enrichment (30%), 132 similar to internal controls (26%) and Arid2Het spermatocytes (Extended Fig. 3c). Therefore, along 133 with centromeric chromatin ARID2 also influences pericentric chromatin organization, albeit at 134 reduced penetrance. 135 136 To understand how ARID2 might restrict centromeric chromatin, we chose to investigate its role 137 in H3T3P regulation. During mitosis, H3T3P is initially detected chromosome-wide, only to be 138 later targeted to centromeres by the phosphatase activity of the CDCA2-PP1 complex, where 139 CDCA2 (also known as Repo-Man) is essential for targeting of the PP1 phosphatase30,31. While 140 the meiotic role of CDCA2 is unknown, it appeared to be highly transcribed at the meiosis-I to 141 spermiogenesis transition8. We therefore monitored CDCA2 localization in metaphase-I 142 spermatocytes by IF. Control metaphase-I spermatocytes exhibited both chromatin associated 143 and cytosolic pools of CDCA2 (Fig. 3c, row 1). In the absence of ARID2, chromatin bound CDCA2 144 was frequently undetectable (51%) (Fig. 3c, row 2). The remainder of the Arid2cKO spermatocytes 145 exhibited either a reduced (4%) or diffuse (26%) CDCA2 targeting to chromatin (Fig. 3c, row 3,4). 146 Interestingly, these defects in CDCA2 targeting appeared consistent with the changes in H3T3P 147 distribution observed in ARID2 deficient metaphase-I spermatocytes (Fig. 3a, c). Therefore, 148 ARID2 might constrain H3T3P by regulating the chromosomal targeting of CDCA2. 149 150 To further investigate this mechanism, we examined BRG1 interacting proteins that were 151 previously identified by immunoprecipitation-mass spectrometry (IP-MS)5. While CDCA2 was 152 absent from this list of candidates, we noticed an association between BRG1 and Protein 153 phosphatase 2 regulatory subunit A alpha (PPP2R1A), a scaffolding subunit of PP2A, whose 154 phosphatase activity is essential for the chromosomal targeting of CDCA232 (Extended Fig. 4a; 155 left). ARID2 and BRG1 co-immunoprecipitations (Co-IP) confirmed the association with PPP2R1A 156 (Fig. 3d; lane 1-2, Extended Fig. 4a; right panel, lane 1). Reverse pulldowns further validated the 157 association with BRG1 but failed to reveal an interaction with ARID2 (Fig. 3d; lane 3, Extended 158 Fig. 4a; right, lane 4). This is likely due to the transient nature of the interaction combined with 159 lower expression of Arid2 relative to Brg1 (Fig. 1a, Extended Fig. 4a; left). In addition to PPP2R1A,

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160 we also detected PP2A, catalytic subunit, PPP2CA33 in the ARID2 Co-IP (Extended Fig. 4a; right, 161 lane 1). These associations prompted us to examine PPP2R1A and PPP2CA localization in 162 metaphase-I spermatocytes in response to the loss of ARID2. While PPP2R1A was detected on 163 chromatin and cytosolically in control metaphase-I spermatocytes, it appeared depleted in 164 majority of the mutant metaphase-I spermatocytes (PLK1-, 71%) (Fig. 3e). Consistent with their 165 interaction, PPP2CA was seen overlapping ARID2 in control metaphase-I spermatocytes. In 166 contrast, PPP2CA appeared diminished with diffuse localization or was absent in Arid2cKO 167 metaphase-I spermatocytes (Extended Fig. 4b). Therefore, the destabilization of the PP2A 168 complex probably explains the chromosome-wide spreading of H3T3P in the absence of ARID2. 169 In fact, the majority of Arid2cKO metaphase-I spermatocytes with reduced PPP2CA levels (83%) 170 displayed H3T3P spreading (Fig. 3f). We conclude that ARID2 impedes H3T3P spreading during 171 metaphase-I by facilitating PP2A mediated targeting of CDCA2 to meiotic bivalents. 172 173 Both H3T3P and H2AT120P are required for the recruitment of the chromosome passenger 174 complex (CPC)4,27,31,34,35, a process necessary for chromosome segregation. We therefore 175 wanted to determine the impact of displaced H3T3P and H2AT120P on CPC localization in 176 Arid2cKO metaphase-I spermatocytes. For this purpose we chose CPC factors known to participate 177 in meiosis, such as Inner centromere protein (INCENP), C (AURKC, meiosis 178 specific) and Aurora kinase B (AURKB)36–39. We first monitored phosphorylated INCENP 179 (pINCENP), considered a member of the CPC localization module40. Similar to previous studies39, 180 pINCENP was detected at centromeres and along the ICA in normal metaphase-I spermatocytes 181 and internal controls from Arid2cKO testis. In contrast, pINCENP was displaced from ICA and 182 centromeres, adopting a predominantly cytosolic localization pattern in mutant metaphase-I 183 spermatocytes (87%) (Fig. 4a). Similar to pINCENP, its meiotic kinase, AURKC was also depleted 184 from chromatin and enriched cytosolically in mutants (70%) compared to internal controls and 185 normal metaphase-I spermatocytes (Fig. 4b). Since AURKC is known to be partially compensated 186 by AURKB during female meiosis36, we were curious to examine AURKB localization in the 187 absence of ARID2. In normal metaphase-I spermatocytes, AURKB was restricted to 188 chromosomes, displaying prominent centromeric enrichment along with diffuse chromatid 189 localization. In contrast, we observed a striking loss of chromosomal AURKB in ARID2 deficient 190 metaphase-I spermatocytes (82%) (Fig. 4c). Therefore, CPC localization is severely impacted in 191 the absence of ARID2. We hypothesize that the mis-regulation of centromeric chromatin 192 contributes to the defect in CPC recruitment.

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193 194 Our studies highlight a crucial requirement for ARID2 and therefore the PBAF complex in 195 reductional male meiosis. This adds to previously identified roles of SWI/SNF in spermatogonial 196 stem cell maintenance and meiotic prophase-I progression5. We show that ARID2 is essential for 197 (i) normal spindle assembly, (ii) PLK1 association with centromeres, (iii) maintaining centromere 198 identity and (iv) the chromosomal targeting of the CPC during metaphase-I, activities that normally 199 promote metaphase exit (Fig. 5). Therefore, it is reasonable to assume that PBAF might function 200 to alleviate the SAC in normal metaphase-I spermatocytes. Interestingly, the loss of ARID2 201 mimics spindle poisons, that inhibit microtubule synthesis and activate a SAC induced metaphase 202 arrest. In contrast to its meiotic role, the PBAF complex prevents aneuploidy during mitosis41 , 203 implying a role in SAC activation. These distinct roles might be attributed to differences in mitotic 204 and meiotic PBAF associations. In spermatocytes we demonstrate that ARID2 interacts with 205 PLK1, a kinetochore associated protein, known to regulate anaphase onset during mitosis42 and 206 oocyte meiosis17 . Therefore, ARID2 might mitigate SAC by facilitating PLK1 recruitment to 207 centromeres. Additionally, the resolution of unattached kinetochores is necessary to overcome 208 SAC. Here, the role of ARID2 in spindle assembly is instructive. Interestingly, ARID2 is abundantly 209 detected at spindle poles in metaphase-I spermatocytes (Extended Fig. 2b). These sites usually 210 represent , a microtubule organizing center, known to be present in spermatocytes43. 211 It is intriguing to speculate whether ARID2 regulates spindle assembly by promoting 212 activity. Alternatively, known mechanisms of spindle formation involving PLK1 and the CPC18– 213 20,44, whose localization are dependent on ARID2 in metaphase-I spermatocytes cannot be ruled 214 out . 215 216 In addition to spindle assembly, PBAF also limits H3T3P and H2AT120P in spermatocytes. In the 217 case of H3T3P, ARID2 appears to employ a conserved mitotic mechanism involving the PP2A 218 regulated chromosomal targeting of CDCA2-PP1, a known H3T3P phosphatase31,32. The fact that 219 this pathway is governed by an association between PBAF and PP2A scaffolding subunit, 220 PPP2R1A is novel. Distinct mechanisms might govern the regulation of H2A120P by ARID2, 221 given that H2AT120P is thought to precede H3T3P. In this regard, the mitotic role of Remodeling 222 and spacing factor1 (RSF1), a member of the ISWI family of ATPases, is informative. Like ARID2, 223 RSF1 is required for the centromeric deposition of PLK145 and promotes centromeric 224 H2AT120P46. The latter activity is achieved by suppressing H2A acetylation. 225

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226 Given that H3T3P and H2AT120P are known to recruit CPC, the eviction of CPC from chromatin 227 featuring ectopic H3T3P and H2AT120P was interesting. Similar defects in the chromatin 228 association of AURKC in oocytes and TFIID during mitosis in response to an excess of H3T3P 229 have been previously reported 27,47. Whether these perturbations result from reduced chromatin 230 accessibility remain unknown. Interestingly, ARID2 prevents the spreading of heterochromatic 231 H3K9me3 in metaphase-I spermatocytes, suggesting a role in promoting chromatin accessibility. 232 233 234 235 236 Acknowledgements

237 We thank Magnuson lab members for helpful comments on manuscript preparation. We thank Dr. Jesse 238 Raab (University of North Carolina, Chapel Hill) for sharing the Arid2 floxed mice. 239 We thank Dr. Michael Lampson (University of Pennsylvania) and Dr. Atilla Töth (Technishe 240 Universität Dresden) for generously providing antibodies. This work was supported by National 241 Institutes of Health grants R01GM101974. 242 243 Statement of competing interests 244 The authors declare no competing financial interests. 245 246 Author contributions 247 D.U.M and T.M conceptualized and designed the project. Data curation and validation done by 248 D.U.M. Writing was performed by D.U.M, reviewed and edited by T.M, D.U.M. Project 249 supervision and funding acquisition done by T.M. 250 251

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252 253 Materials and Methods: 254 Generation of Arid2 conditional deletion and genotyping: 255 A knockout first allele of Arid2 (Arid2tm1a(EUCOMM)Wtsi) was obtained from EUCOMM48 and the mice 256 were generated at the regional mutant mouse resource and research center (MMRRC; Stock 257 number: 036982-UNC; www.mmrrc.org) at the university of North Carolina at Chapel Hill. The 258 lacZ trap was flipped out to generate Arid2 floxed mice (Arid2tm1c(EUCOMM)Wtsi) that were 259 maintained on a mixed genetic background. Conditional knockout was achieved using Stra8-Cre 260 (activated only in males at P3) 13. Arid2fl/fl females were crossed to Arid2fll+; Stra8-CreTg/0 males to 261 obtain Arid21flΔ;Stra8-CreTg/0 (Arid2cKO) , Arid21flΔ;Stra8-CreTg/0 (Arid2Het) and Arid2fl+ (Arid2WT) 262 males. Genotyping primers used in this study include: Arid2fl+ alleles - (F)- 5’ - 263 CTGCTTAGCCCAAAGGTGTC -3’ ; (R) 5’ - GACAGTGACTTCAGCTGACC -3’, the excised allele 264 (Δ)- forward primer used above in combination with (R) 5’ -CTGAGCCCAGGTGTTTTTGT-3’ and 265 Stra8-Cre - (F) 5’ -GTGCAAGCTGAACAACAGGA-3’, (R) 5’ -AGGGACACAGCATTGGAGTC-3’. 266 All animal work was carried out in accordance with approved IACUC protocols at the University 267 of North Carolina at Chapel Hill. 268 269 Histology: 270 Testes and cauda epididymides from adult Arid2WT and Arid2cKO mice were fixed and stained as 271 described previously49. Periodic acid-Schiff (PAS), hematoxylin and eosin staining, and sectioning 272 of tissue was performed by the animal histopathology core at the University of North Carolina at 273 Chapel Hill. Seminiferous tubule staging and cell type identification were based on methods 274 outlined previously 50,51. 275 276 Immunofluorescence staining: 277 Immunofluorescence (IF) was conducted on testes cryosections, squashes and spermatocyte 278 spreads obtained from juvenile (P22 - P27) and adult Arid2WT, Arid2Het and Arid2cKO mice. IF for 279 metaphase spermatocytes were performed on both testes cryosections and seminiferous tubule 280 squashes. We ensured that IF results were consistent across preparations. Testis cryosections 281 (8-10 μm thick) were prepared and immunostained as previously described5. Identical method 282 was used to immunolabel seminiferous tubule squashes, prepared by previously described 283 technique52 with one minor modification. To ensure that the squashes adhered to the surface of 284 the glass slide prior to coverslip removal, inverted slides were subjected to downward pressure

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285 for 20 minutes by placing above them an eppendorf rack bearing added weight in the form of an 286 aluminum heating block or a filled 500 ml glass bottle. “3D-preserved” prophase-I spermatocyte 287 spreads were prepared and immunolabeled using previously described methods 5,25. Entire list 288 of primary and secondary antibodies used for IF are provided in supplemental information 289 (Extended table. 1). Images were acquired using Zeiss AxioImager-M2 and Leica Dmi8 290 fluorescent microscopes. Z-stacks were deconvoluted by Axiovision (AxioImager images) or 291 Huygens (Leica Dmi8 images) software. Image processing and pixel quantification were 292 performed with Fiji53. 293 294 Preparation of metaphase spreads: 295 DAPI stained metaphase chromosome spreads were prepared from P19 - P27 and adult Arid2WT, 296 Arid2Het and Arid2cKO testes, using a previously described technique54. 297 298 Preparation of nuclear lysates for immunoprecipitation: 299 Nuclear lysates were obtained from spermatocyte enriched populations isolated from 3-week-old 300 males by methods described previously 55,56. Protein extracts for immunoprecipitation were 301 isolated from nuclei using a high salt extraction method5. 302 303 Co-Immunoprecipitation (Co-IP): 304 Co-IP were performed exactly as described previously5 with minor modifications. These include 305 (i) Incubation of antibodies with nuclear lysates (500 - 600 μg/IP) prior to capture of antibody- 306 antigen complexes with magnetic protein A beads for 3 hours over a rotator at 4 C, (ii) Milder IP 307 wash conditions involving two IP buffer washes, followed by sequential washes once in high salt 308 wash buffer, low salt wash buffer and final wash buffer. 309 310 Preparation of acid extracted histones: 311 Histones were extracted from spermatogenic cells obtained from P23 and P27 Arid2WT, Arid2Het 312 and Arid2cKO mice using acid extraction protocol described previously 57. 313 314 Western blotting: 315 Protein samples were separated by polyacrylamide gel electrophoresis and immunoblotted as 316 described previously5. Sample loading was assessed by staining blots with REVERTTM total

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317 protein reagents (LI-COR). Blots were scanned on a LI-COR Odyssey imager. All the antibodies 318 used in this study and their corresponding dilutions are listed (Extended table. 1). 319 320 Data sets analyzed: 321 RNA-seq data from purified male germ cells was previously generated (GEO accession: 322 GSE35005)8. PPP2R1A association with SWI/SNF was obtained from unpublished data 323 generated using BRG1 IP-MS reported previously5. 324

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457 54. Meredith, R. A simple method for preparing meiotic chromosomes from mammalian 458 testis. Chromosoma 26, 254–258 (1969). 459 55. Chang, Y. F., Lee-Chang, J. S., Panneerdoss, S., MacLean, J. a. & Rao, M. K. Isolation 460 of Sertoli, Leydig, and spermatogenic cells from the mouse testis. Biotechniques 51, 341– 461 344 (2011). 462 56. Mu, W., Starmer, J., Fedoriw, A. M., Yee, D. & Magnuson, T. Repression of the soma- 463 specific transcriptome by Polycomb-repressive complex 2 promotes male germ cell 464 development. Genes Dev. 28, 2056–2069 (2014). 465 57. Shechter, D., Dormann, H. L., Allis, C. D. & Hake, S. B. Extraction, purification and 466 analysis of histones. Nat. Protoc. 2, 1445–1457 (2007). 467 468

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469 Figure Legends 470 471 Figure 1. The loss of ARID2 results in a late meiosis-I arrest. (a) mRNA expression profile of 472 SWI/SNF subunits in purified germ cell populations8. (b) Adult testis cryosection immunolabelled 473 for ARID2 (magenta), gH2Ax (yellow) and counterstained with DAPI (blue), scale bar: 20 μm. 474 Magnified views of representative cell types are displayed below, scale bar: 5 μm. 475 Magnification:40x. (c) Arid2WT and Arid2cKO testis mounts. Average testis mass (g) and standard 476 error obtained from multiple measurements (n) are indicated. p-value calculated using students t- 477 test. Scale bar: 500 μm. (d) 12-week-old Arid2WT and Arid2cKO PAS-stained testes sections . 478 Magnified view of Arid2cKO metaphase spermatocytes are displayed. Scale bar: 50 μm, 479 magnification: 40x. (e) Proportions of prophase-I spermatocytes (pSpc) and spermiogenic cell 480 populations quantified from 12-week-old Arid2WT and Arid2cKO PAS-stained testes sections. 481 Number of tubules (n) examined for each genotype are indicated. (a,b,d) Spg: Spermatogonia, 482 Lep: Leptotene spermatocyte, Pach/P: Pachytene spermatocyte, Zyg/Z: Zygotene spermatocyte, 483 M: Metaphase-I spermatocyte, rSt: round spermatid and eSt: elongated spermatid. 484 485 Figure 2. ARID2 influences spindle assembly and PLK1 association with kinetochore. (a,b) 486 Metaphase-I spermatocytes from control and Arid2cKO testes cryosections immunolabelled for (a) 487 H3S10P (green) and β-Tubulin (magenta), (b) PLK1 (green), CENPA (magenta) and 488 counterstained with DAPI (blue). Arrowheads denote cell of interest and panel insets highlight 489 centromeres. (a,b) Scale bars: 5 μm, magnification:100x. Internal control (Spindle+/PLK1+) and 490 mutant metaphase-I spermatocytes (Spindle-/PLK1-) were imaged from same section. Total 491 number of metaphase-I spermatocytes scored (n) from Arid2cKO seminiferous tubules and 492 proportion (%) of mutant cells are indicated. (c) ARID2 (top) and BRG1 (bottom) Co-IP. Red 493 arrowhead indicates interacting protein, yellow arrow denotes non-specific streak. 494 495 Figure 3. ARID2 regulates centromeric chromatin in metaphase-I spermatocytes. (a-c) 496 Control and Arid2cKO metaphase-I spermatocyte squashes immunolabelled for PLK1 (green) and 497 (a) H3T3P (magenta), (b) H2AT120P (magenta) and (c) CDCA2 (magenta). DNA stained with 498 DAPI. Scale bar: 5 μm, magnification: 100.8x. (d) ARID2, BRG1 and PPP2R1A Co-IP’s. Red 499 arrowheads indicate interacting proteins. Numbers at bottom denote sample lanes. (e,f) Control 500 and Arid2cKO metaphase-I spermatocytes immunolabelled for (e) PPP2R1A (magenta) and PLK1 501 (green), (f) H3T3P (magenta) and PPP2CA (green). Scale bar: 5 μm, magnification: 100.8x. (a-c,

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502 e-f) Total number of metaphase-I spermatocytes scored (n) from Arid2cKO seminiferous tubules 503 and proportion (%) of each abnormal category are indicated. 504 505 Figure 4. ARID2 influences the localization of the meiotic chromosome passenger 506 complex. (a,b) Arid2WT and Arid2cKO metaphase-I spermatocyte squashes immunolabelled for 507 PLK1 (green) and (a) pINCENP (magenta), (b) AURKC (magenta). Internal controls (white 508 arrowhead) and mutants (yellow arrowhead) are indicated. (c) Arid2Het and Arid2cKO metaphase-I 509 spermatocyte squashes immunolabelled for AURKB (magenta) and ARID2 (green). DNA stained 510 with DAPI. (a-c) Total number of metaphase-I spermatocytes scored (n) from Arid2cKO 511 seminiferous tubules and proportion (%) of mutant cells are indicated. Scale bar: 5 μm, 512 magnification: 100.8x. 513 514 Figure 5. Model describing the role of ARID2 in spermatocyte cell division. ARID2 is 515 required for normal spindle assembly and chromosome segregation in metaphase-I 516 spermatocytes. The association with essential cell division factors such as PLK1 and PP2A 517 implicates PBAF in the regulation of metaphase-I exit. In the absence of ARID2, spermatocytes 518 arrest at metaphase-I, characterized by the lack of spindle, absence of kinetochore associated 519 PLK1, chromosome-wide distribution of H3T3P and H2AT120P and mis-localization of meiotic 520 chromosome passenger complex (CPC). The mis-targeting of H3T3P in the absence of ARID2 521 occurs due to destabilization of PP2A which results in the reduced chromosomal occupancy of 522 H3T3P phosphatase, CDCA2-PP1. CEN: Centromere 523 524 525 526

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527 Extended Data 528 529 Extended figure legends 530 531 Extended figure 1. Characterization of ARID2 during spermatogenesis. (a) Western blot 532 displaying ARID2 knockout in spermatogenic cells obtained from juvenile (P21), adult (12-week) 533 Arid2WT and Arid2cKO testes. Nucleolin (NCL) was used as nuclear loading control. (b) H&E 534 stained paraffin sections of cauda epididymides obtained from adult Arid2WT and Arid2cKO mice. 535 Scale bar: 50 μm, magnification: 25x. (c) Meiotic prophase-I profiles obtained from juvenile 536 Arid2Het and Arid2cKO spermatocyte spreads. Prophase-I substages were identified by SYCP3 537 and γH2Ax immunostaining. (d) ARID2 (magenta) and BRG1 (green) localization in metaphase- 538 I and anaphase-I spermatocytes. DNA counterstained with DAPI. Scale bar: 5 μm, 539 magnification: 100x. 540 541 Extended figure 2. Features of Arid2cKO spermatocytes. (a) DAPI-stained Arid2Het and 542 Arid2cKO metaphase spreads, scale bar: 5 μm, magnification:100x. (b, c) Metaphase-I 543 spermatocytes from Arid2Het and Arid2cKO testes cryosections immunolabelled for (a) β-Tubulin 544 (magenta) and ARID2 (green), (c) PLK1 (green) and ARID2 (magenta) with quantification (right) 545 of ARID2 signal (y-axis) from Arid2Het and Arid2cKO internal control (yellow asterisk, PLK1+) and 546 mutant (white asterisk, PLK1) metaphase-I spermatocytes. Number of spermatocytes quantified 547 under each category (x-axis) are indicated (n). (d) Arid2Het and Arid2cKO diplotene 548 spermatocytes immunolabeled for HORMAD1 (magenta), PLK1 (green) and ARID2 (white). All 549 immunostained preparations were counterstained with DNA. Scale bars: 5 μm, magnification: 550 100.8x 551 552 Extended figure 3. Effect of ARID2 on centromeric and pericentric histone modifications. 553 (a, b) Western blots on acid extracted histones obtained from P23, P27 control and Arid2cKO 554 spermatogenic cells, depicting abundance of (a) H3T3P (top), H3K9me3 (bottom), and (b) 555 H2AT120P. Total protein from each sample are displayed. (c) Control and Arid2cKO metaphase-I 556 spermatocytes immunolabelled for H3K9me2 (magenta) and PLK1 (green). DNA stained with 557 DAPI. Total number of metaphase-I spermatocytes scored from Arid2cKO seminiferous tubules 558 are indicated (n). Scale bar: 5 μm, magnification: 100.8x.

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559 560 Extended figure 4. ARID2 associates with PP2A. (a) Table (left) summarizing PPP2R1A 561 interaction with BRG1 previously identified by IP-MS and ARID2, PPP2R1A Co-IP’s (right). Red 562 arrowheads indicate interacting proteins, yellow arrow denotes non-specific streak. Numbers at 563 bottom denote sample lanes. (b) Control and Arid2cKO metaphase-I spermatocytes 564 immunolabelled for ARID2 (magenta) and PPP2CA (green). Total number of metaphase-I 565 spermatocytes scored from Arid2cKO seminiferous tubules are indicated (n). Scale bar: 5 μm, 566 magnification: 100.8x 567 568 569

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570 Extended table 1. List of antibodies Antibody Vendor Application Rabbit anti-ARID2 Thermofisher (PA5-35857) IF (1:100) IP (10 ug) WB (1:1000) Mouse anti- BRG1 (G-7) SantaCruz Biotech (sc- 17796) IF (1:100) WB (1:1000) Rabbit anti- BRG1 Abcam (ab110641) IP (7 μg) IF (1:500) Mouse anti- γH2Ax Millipore (05-636) IF (1:1000) Rabbit anti NCL (Nucleolin) Bethyl (A300-711A) WB (1:5000) Mouse anti- SYCP3 Abcam (ab97672) IF (1:500) Rabbit anti- γH2Ax Cell signaling (9718) IF (1:1000) Rabbit anti- H3S10P Millipore (06-570) IF (1:500) Mouse anti β-TUBULIN DHSB (E7) IF (1:25) Rabbit anti- CENPA Abcam (ab33565) IF (1:25) Mouse anti- PLK1[35-206] Abcam (ab17056) IF (1:200) WB (1:1000) Guinea pig anti- HORMAD1 Gift from Dr. Atilla Töth, TU IF (1:500) Dresden Rabbit anti- H3T3P Active Motif (39153) IF (1:100) WB (1:1000) Rabbit anti- H2AT120P Active Motif (39392) IF (1:100) WB (1:1000) Rabbit anti- H3K9me3 Abcam (ab8898) IF (1:500) WB (1:1000) Rabbit anti- CDCA2 Sigma (HPA030049) IF (1:100) Rabbit anti- PPP2R1A Proteintech (15882-1-AP) IF (1:100) IP (1.6 μg – 8 μg) WB (1:800) Mouse anti- PPP2CA DHSB (CPTC-PP2A-4) IF (1:10) WB (1:20) Rabbit anti- phosphoINCENP Gift from Dr. Michael Lampson, IF (1:500) University of Pennsylvania Rabbit anti – AURKC Bethyl (A300-BL1217) IF (1:30) Mouse anti- AURKB BD biosciences (611082) IF (1:30) Secondary antibodies Goat anti-mouse, Alexa fluor 488 ThermoFisher (A-11029) IF (1:500) Goat anti-mouse, Alexa fluor 568 ThermoFisher (A-11031) IF (1:500) Goat anti-rabbit, Alexa fluor 568 ThermoFisher (A-11036) IF (1:500) Goat anti- rabbit, Alexa fluor 488 ThermoFisher (A-11008) IF (1:500) Goat anti- guinea pig, Alexa fluor 568 ThermoFisher (A-11075) IF (1:500) Goat anti- rabbit, Alexa fluor 647 ThermoFisher (A-21245) IF (1:500) IRdye 680LT Goat anti-rabbit LI-COR (926-68021) WB (1:20,000) IRdye 680LT Goat anti-mouse LI-COR (925-68020) WB (1:20,000) IRdye 800CW Goat anti-mouse LI-COR (926-32210) WB (1:10,000)

21 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.28.066647; this version posted April 28, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Menon Fig 1 a b Gene.Symbol Arid1aArid1a Arid1b ARID2 Arid1b γH2Ax Arid2 150150 Arid2 DAPI Pbrm1Pbrm1 Smarca4 Smarca4Gene.Symbol Arid1a

100 Arid1b

100TPM Arid2 Pbrm1 Smarca4 TPM 5050

00 Pre.SpgA SpgA SpgB Lep Pach rST eST Cell_Identity rSt eSt SpgASpgB Lep Pach Pre-SpgA c Pach rST eSTp < 0.001 Cell_Identity

Mass (g): 128.7 ± 2.8 55.2 ± 2.1 ARID2 Arid2WT Arid2cKO DAPI

Pre-Lep Zyg Pach rSt 12-wk testis n=9 n=6 d e

Arid2WT Arid2WT (n=17)

cKO 120&"! Arid2 (n=24) eSt 100&!!

P 80%! Z 60$!

40#! cKO Arid2 20"! Average count/tubule Average M P 0! M pSpc rSt eSt Cell Identity Z Metaphase - I arrest

Stage XII bioRxiv preprint doi: https://doi.org/10.1101/2020.04.28.066647; this version posted April 28, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Menon Fig 2 a c H3S10P + β TUBULIN

WT IP: ARID2 IgG Input ARID2 Arid2 β Tubulin H3S10P

PLK1 Western Blot (n=220) + IP: BRG1 Input IgG

cKO Spindle BRG1 Arid2

PLK1

Spindle- 52 % b PLK1 + CENPA + DNA Het Arid2 CENPA PLK1 DNA

(n=133) PLK1+ cKO Arid2

PLK1- 60 % a c Menon Fig 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.28.066647; this version posted April 28, 2020. The copyright holder for this preprint (which H3T3Pwas not certified by peer review) is the author/funder, who has granted bioRxivCDCA2 a license to display the preprint in perpetuity. It is made PLK1 available under aCC-BY-ND 4.0 InternationalPLK1 license. WT DNA Het DNA Arid2 Arid2

H3T3P DNA CDCA2 DNA

Int. Cont 10 % Absent 51 % (n=129) (n=194) cKO cKO

60 % 4 % Arid2

Spread Arid2 Low

Low 30 % Diffuse 26 % b d

H2AT120P PLK1

Het IP: ARID2 BRG1 PPP2R1AInput IgG DNA BRG1 (Low Exp) Arid2 BRG1 Western Blot H2AT120P DNA (High Exp) ) ARID2 (n=147 PPP2R1A cKO

62 % 1 2 3 4 5

Arid2 Spread e f PPP2R1A H3T3P PLK1 PPP2CA Het Het DNA DNA Arid2 Arid2

PPP2R1A DNA PPP2CAPP2AC H3T3PH3T3P (n=106) (n=78) cKO cKO

71 % 83 % Arid2 Arid2 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.28.066647; this version posted April 28, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Menon Fig 4 a pINCENP PLK1 WT DNA Arid2

pINCENP DNA (n=123)

cKO 87 % Arid2 b AURKC PLK1

WT DNA Arid2

AURKC DNA (n=226) cKO 70 % Arid2 c AURKB ARID2

Het DNA Arid2

AURKB DNA (n=50) cKO

Arid2 82 % bioRxiv preprint doi: https://doi.org/10.1101/2020.04.28.066647; this version posted April 28, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

WT Menon Fig 5 Arid2

PLK1 H2AT120P CEN Metaphase - I PBAFARID2 H3T3P PP2A CPC CDCA2 PP1

CPC H2AT120P H3T3P Metaphase-I CEN Exit Bivalent PLK1

Chromosome Seggregation

Arid2cKO

PLK1 CPC Metaphase - I PBAF H2AT120P CEN H3T3P PP2A

pCDCA2PP1

H2AT120P Metaphase -I H3T3P CEN CPC Arrest Bivalent PLK1

Abnormal Chromosome Organization

Aberrant Spindle bioRxiv preprint doi: https://doi.org/10.1101/2020.04.28.066647; this version posted April 28, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Menon Ext Fig 1 a b WT cKO Cauda Epididymis Arid2 Arid2

ARID2

NCL P21 Testes

Arid2WT Arid2cKO

ARID2 Nuclear extracts

12 wk Testes NCL

c d DNA ARID2 BRG1 MERGE Meiotic prophase-I profile 100% *

75% lepLeptonema

zyg Metaphase - I 50% Zygonema pach Percentage Pachynema

25% dip Percentage Diplonema * 0% * Het cKO Anaphase - I Arid2Arid2Het Arid2Arid2KO Menon Ext Fig 2 a bioRxiv preprint doi: https://doi.org/10.1101/2020.04.28.066647; this version posted Aprilb 28, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. β TUBULIN + ARID2 + DNA Het Pro metaphase - I Metaphase - I Metaphase - II

XY Arid2

Het ARID2 Arid2

cKO + cKO XY Spindle Arid2 Arid2

Spindle- c PLK1 + ARID2 + DNA n=44

Het 200 2 n=61

Arid2 n=8 150 ARID2 100

cKO * *

ARID2 signal/pixel 50 Arid2

* 0 * PLK1+ PLK1- Arid2Het Arid2cKO d MERGE HORMAD1 PLK1 ARID2 DNA Het Arid2 cKO Arid2 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.28.066647; this version posted April 28, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a licenseMenon to display theExt preprint Fig in perpetuity. 3 It is made available under aCC-BY-ND 4.0 International license. a c P23 P27

WT Het cKO Het cKO H3K9me3 PLK1

Arid2 Arid2 Arid2 Arid2 Arid2 Het DNA

15 kDa Arid2 H3K9me3 H3T3P

25 kDa

Int. Cont 26 % (n=124)

Total Histone Total 10 kDa cKO Arid2 15 kDa Peri-centric 30 % H3K9me3

25 kDa

Spread 38 %

10 kDa Total Histone Total b P23 P27

WT cKO WT cKO

Arid2 Arid2 Arid2 Arid2

15 kDa H2AT120P

25 kDa

10 kDa Total Histone Total bioRxiv preprint doi: https://doi.org/10.1101/2020.04.28.066647; this version posted April 28, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license.

Menon Ext Fig 4 a Co-Immunoprecipitation

BRG1 IP-MS from 3 wk old testes IP: ARID2 IgG Input PPP2R1A Protein # Unique Peptide % Coverage BRG1

PPP2R1A 3 9.3 ARID2 Western Blot

PPP2R1A

PPP2CA

1 2 3 4 b ARID2 + PPP2CA + DNA Het Arid2 PPP2CA DNA (n=73) cKO Arid2