ARTICLE Received 28 Jan 2015 | Accepted 7 Jul 2015 | Published 18 Aug 2015 DOI: 10.1038/ncomms9017 OPEN CRL4–DCAF1 ubiquitin E3 ligase directs protein phosphatase 2A degradation to control oocyte meiotic maturation Chao Yu1, Shu-Yan Ji1,*, Qian-Qian Sha1,*, Qing-Yuan Sun2 & Heng-Yu Fan1 Oocyte meiosis is a specialized cell cycle that gives rise to fertilizable haploid gametes and is precisely controlled in various dimensions. We recently found that E3 ubiquitin ligase CRL4 is required for female fertility by regulating DNA hydroxymethylation to maintain oocyte survival and to promote zygotic genome reprogramming. However, not all phenotypes of CRL4-deleted oocytes could be explained by this mechanism. Here we show that CRL4 controls oocyte meiotic maturation by proteasomal degradation of protein phosphatase 2A scaffold subunit, PP2A-A. Oocyte-specific deletion of DDB1 or DCAF1 (also called VPRBP) results in delayed meiotic resumption and failure to complete meiosis I along with PP2A-A accumulation. DCAF1 directly binds to and results in the poly-ubiquitination of PP2A-A. Moreover, combined deletion of Ppp2r1a rescues the meiotic defects caused by DDB1/DCAF1 deficiency. These results provide in vivo evidence that CRL4-directed PP2A-A degradation is physiologically essential for regulating oocyte meiosis and female fertility. 1 Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University, Hangzhou 310058, China. 2 State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to H.-Y.F. (email: [email protected]). NATURE COMMUNICATIONS | 6:8017 | DOI: 10.1038/ncomms9017 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9017 o produce fertilizable eggs with the appropriate number In this study we investigate the physiological functions and of chromosome sets, oocyte meiotic progression is biochemical mechanisms of CRL4DCAF1 in regulating Tprecisely regulated1. In ovaries of adult female mammalian oocyte meiotic maturation. DDB1 or DCAF1 mammals, oocytes are arrested at the diplotene stage of deletion causes delayed meiotic resumption and failure to prophase I, which is also called germinal vesicle (GV) stage2. complete meiosis I in fully grown oocytes. The PP2A-A subunit A luteinizing hormone surge at oestrus triggers oocyte meiotic is accumulated in DDB1- or DCAF1-deleted oocytes, which resumption in preovulatory follicles, followed by chromosome inhibits cohesin removal and homologous chromosome alignment and spindle organization at prometaphase I separation during meiosis I. Biochemistry assays shows (Pro-MI)3,4. Accurate homologous chromosome separation is that CRL4DCAF1 binds to PP2A-A and targets it for poly- achieved by cohesion removal after all chromosome pairs are ubiquitination and proteasome degradation. Our results aligned along the spindle equatorial plane5,6. Subsequently, demonstrate for the first time that CRL4DCAF1 Ub E3 ligase is oocytes extrude a first polar body (PB1) and are arrested at essential for promoting meiotic cell cycle progression by targeting metaphase II (MII) to await fertilization. Dysregulated meiotic PP2A for degradation in mouse oocytes. progression results in aneuploidy and embryonic development abnormality, which contributes to early abortion and female infertility7. Results Protein phosphatase 2A (PP2A) is a known cell cycle regulator CRL4DCAF1 complexes are essential for oocyte meiosis.To in both somatic cells and oocytes. PP2A consists of a scaffold A investigate the functions of CRL4DCAF1 complexes during oocyte subunit, a catalytic C subunit and a regulatory B subunit8. meiotic maturation, we selectively deleted Ddb1 or Dcaf1 in Previous studies showed that increased PP2A activity oocytes by using previously reported conditional knockout mouse inhibited oocyte GV breakdown (GVBD) by counteracting models, Ddb1fl/fl;Gdf9-Cre and Dcaf1fl/fl;Gdf9-Cre (hereafter CDK1 activation9. In contrast, genetically deleting Ppp2r1a in referred to as Ddb1oo À / À and Dcaf1oo À / À )19. oocytes, a gene encoding for the major isoform of the PP2A-A Immunohistochemistry results confirmed that DDB1 and subunit, facilitated GVBD, which suggested that PP2A functioned DCAF1 were specifically deleted in oocytes from Ddb1oo À / À as an inhibitor of meiotic resumption10. In addition, PP2A is and Dcaf1oo À / À mice, respectively (Supplementary Fig. 1). required to prevent precocious separation of sister We then investigated the meiotic maturation of DDB1- and chromatids during oocyte meiosis I10,11. During meiotic DCAF1-deficient oocytes. In wild-type (WT) females, 95% of the division I, sister chromatids are attached to one another by oocytes within preovulatory follicles resumed meiosis and cohesin complexes when homologous chromosomes form underwent GVBD at 4 h after human chorionic gonadotropin tetrads. During the MI-to-AI transition, cohesins on (hCG) administration (Fig. 1a, white arrowheads). However, in chromosome arms are phosphorylated and then cleaved by the ovaries of Ddb1oo À / À and Dcaf1oo À / À mice, most separase, while centromeric cohesins are protected by a PP2A- oocytes within preovulatory follicles had intact GVs (Fig. 1a, shugoshin complex through dephosphorylation5,6,11,12. Although black arrowheads) at 4 h post-hCG injection. Only 32.9 and 8.7% PP2A controls meiotic resumption and ensures accurate of these oocytes underwent GVBD (Fig. 1b). We superovulated chromosome separation, little is known regarding how PP2A WT, Ddb1oo À / À and Dcaf1oo À / À females and collected protein stability is regulated during cell cycles. oocytes at 16 h post-hCG injection. GVBD occurred in 83% of Protein ubiquitin (Ub) E3 ligases, in particular, play key roles Ddb1oo À / À oocytes, but none of these oocytes extruded PB1s. during both meiotic and mitotic cell cycle progression by More severe defects were observed for Dcaf1oo À / À oocytes, triggering specific proteins degradation. For example, an as only 52.6% of the ovulated oocytes underwent GVBD and anaphase promoting complex (APC) initiates the metaphase– none released a PB1 (Fig. 1c,d). Cultured Ddb1oo À / À and anaphase transition by inducing the degradation of cyclin B and Dcaf1oo À / À oocytes also had significantly low GVBD rate and securin13. Cullin-ring ligase-1 (CRL1) promotes early mitotic extruded no PB1s in vitro (Fig. 1e–g and Supplementary Fig. 2a). inhibitor 1 degradation and facilitates oocyte meiotic resumption For the reason that DDB1 protein is more stable than DCAF1, and progression to MI14. However, it remains unclear whether Dcaf1oo À / À oocytes exhibited stronger phenotypes than PP2A is also regulated by the ubiquitination–proteasome Ddb1oo À / À oocytes19. pathway as part of the biochemical network that controls the In addition, Ddb1oo À / À and Dcaf1oo À / À oocytes that cell cycle. resumed meiosis had abnormal spindles and misaligned CRL4 Ub E3 ligase is evolutionally conserved from yeasts to chromosomes (Supplementary Fig. 2b, arrows). When humans. Its core components include cullin 4A or B as a scaffold, superovulated Ddb1oo À / À females were mated with fertile adult damaged DNA-binding protein 1 (DDB1) as a linker, and the males, 2-cell embryos were not found in their oviducts at 44 h ring finger protein ROC1/2 (refs 15,16). By selectively forming post-hCG injection (Supplementary Fig 2c,d). protein complexes with its more than 90 substrate adaptors We then monitored the meiotic maturation of WT and known as DDB1-CUL4 associated factors (DCAFs), CRL4 DDB1-deleted oocytes in vitro using live imaging. mRNAs regulates a wide range of cellular processes17,18. We previously encoding for green fluorescent protein (GFP)-tubulin and generated oocyte-specific Ddb1 and Dcaf1 knockout mice and RFP-H2B were co-injected into oocytes to demonstrate spindle found that CRL4 in complex with one of its substrate assembly and chromosome organization. Compared with WT adaptors, DCAF1, also known as VPRBP, regulated the oocytes, GVBD was delayed in DDB1-deleted oocytes. Even after hydroxymethylation of genomic DNA to maintain oocyte GVBD, these oocytes failed to emit a PB1 and did not complete survival and to promote zygotic genome reprogramming after meiosis I. Although spindles were formed in these oocytes, fertilization19,20. Interestingly, although these knockout mice condensed chromosomes did not align at the equatorial plane could ovulate in response to exogenous gonadotropin when they (Supplementary Fig. 3). were young, most of their ovulated oocytes had abnormal In DDB1- or DCAF1-deleted oocytes that resumed meiosis morphologies, as characterized by the presence of GVs and the within 4 h, lagged chromosomes were frequently observed, as in absence of PB1s. This suggested that, in addition to maintaining matured oocytes in vivo (Fig. 1e and Supplementary Fig. 2a). the survival of dormant oocytes, CRL4DCAF1 was a crucial Homologous chromosome separation and PB1 emission (PBE) regulator of meiotic maturation in fully grown oocytes. were not observed in these oocytes (Fig. 1e). 2 NATURE COMMUNICATIONS | 6:8017 | DOI: 10.1038/ncomms9017 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9017 ARTICLE a WT Ddb1fl/fl;G-Cre Dcaf1fl/fl;G-Cre b 120 *** 100 *** 80 60 40 hCG 4 h GVBD rate (%) GVBD rate 20 0 fl/fl fl/fl WT Ddb1 Dcaf1 G-Cre G-Cre c fl/fl fl/fl d WT Ddb1 ;G-Cre Dcaf1 ;G-Cre GV Pro-MI MII 120 n=65 n=54 n=57 100 80 60 hCG 16 h 40 Oocyte stage (%) 20 0 fl/fl fl/fl e fl/fl fl/fl WT Ddb1 Dcaf1 WT Ddb1 ;G-Cre Dcaf1 ;G-Cre G-Cre G-Cre f WT (n=207) Ddb1fl/fl;G-Cre (n=288) Dcaf1fl/fl;G-Cre (n=94) 120 100 *** *** *** Culture 8 h 80 *** 60 Percentage (%) Percentage 40 20 0 Culture 4 h 14 h -Tubulin GVBD PBE α / g WT Culture 9.5 h DNA Ddb1fl/fl;G-Cre 120 100 80 n=93 60 40 n=61 GVBD rate (%) GVBD rate Culture 12 h PB1 20 0 Time (h) 2 4 8 10 Figure 1 | DDB1 or DCAF1 deletion in mouse oocytes causes meiotic maturation defects.