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CRL4 Complex Regulates Mammalian Oocyte Survival and Reprogramming by Activation of TET Proteins

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Science  20 Dec 2013:
Vol. 342, Issue 6165, pp. 1518-1521
DOI: 10.1126/science.1244587

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Ubiquitin Fertility Insurance

The female mammal's reproductive lifespan is determined by a pool of ovarian primordial follicles that are generated early in life. Yu et al. (p. 1518) found that in mice, the ubiquitin E3 ligase complex CRL4 is essential for oocyte survival within primordial follicles and for development after fertilization. CRL4 binds to and activates an adaptor protein that mediates ubiquitination, but if any component is deleted, the genes required for oocyte maintenance and early embryo development are silenced and the female mice become infertile.

Abstract

The duration of a woman’s reproductive period is determined by the size and persistence of a dormant oocyte pool. Specific oocyte genes are essential for follicle maintenance and female fertility. The mechanisms that regulate the expression of these genes are poorly understood. We found that a cullin-ring finger ligase-4 (CRL4) complex was crucial in this process. Oocyte-specific deletion of the CRL4 linker protein DDB1 or its substrate adaptor VPRBP (also known as DCAF1) caused rapid oocyte loss, premature ovarian insufficiency, and silencing of fertility maintaining genes. CRL4VPRBP activates the TET methylcytosine dioxygenases, which are involved in female germ cell development and zygote genome reprogramming. Hence, CRL4VPRBP ubiquitin ligase is a guardian of female reproductive life in germ cells and a maternal reprogramming factor after fertilization.

In the mammalian ovary, primordial follicles are generated early in life and form a reservoir of female germ cells. To ensure a sufficiently long reproductive period, some primordial follicles must survive in a resting state for months (mice) or decades (humans) (1). However, the molecular mechanisms that control the balance between primordial follicle survival and loss are not well known.

The cullin family proteins assemble as many as 400 cullin-ring finger ligase (CRL) complexes that regulate diverse cellular pathways, but none have been functionally analyzed in oocytes. Cullin 4 (CUL4) utilizes damaged DNA binding protein–1 (DDB1) as a linker to interact with a subset of DDB1-cullin–associated factors (DCAFs), which act as substrate receptors (2, 3). Viral protein R (VPR)–binding protein, VPRBP (DCAF1), was an important CRL4 adaptor in these processes.

The CRL4 components CUL4A/B, ring of cullin–1 (ROC1), and DDB1 were highly expressed in mouse oocytes (figs. S1 and S2). We generated oocyte-specific and developmental stage–specific Ddb1 knockout mice by crossing Ddb1fl/fl mice with Ddx4-Cre, Gdf9-Cre, and Zp3-Cre transgenic mice (fig. S3A). For all resulting mouse strains, females were infertile (fig. S3B). In Ddb1fl/–;Ddx4-Cre mice, DDB1 was deleted in oocytes on postnatal days 1 to 3 (PDs 1 to 3) (fig. S3C). Ddb1fl/–;Ddx4-Cre ovaries at PD1 contained oocytes at numbers comparable to those in wild-type (WT) controls, whereas oocyte loss and apoptosis were notable at PD3 (Fig. 1, A to C, and fig. S3, D and E). All oocytes were lost in these mice by young adulthood (fig. S3F).

Fig. 1 CRL4VPRBP in oocyte is required for primordial follicle maintenance.

(A) MVH IHC staining in WT and Ddb1fl/–;Ddx4-Cre ovaries. Scale bars, 30 μm. (B) Oocyte numbers in WT and Ddb1fl/–;Ddx4-Cre (Ddb1;D-Cre) ovaries at PDs 1 to 3. Error bars indicate SEM. (C) Quantification of apoptotic oocytes in WT and Ddb1fl/–;Ddx4-Cre ovaries in fig. S3E. (D) H&E staining and MVH IHC of ovaries from 12-week-old mice with the indicated genotypes. Scale bar, 250 μm.

The ovaries of 6-week-old Ddb1fl/fl;Gdf9-Cre females showed no histological abnormalities (fig. S4, A and B). However, for Ddb1fl/fl;Gdf9-Cre females older than 8 weeks, the ovaries were smaller than controls’ (fig. S4A). Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for MVH (oocyte marker) and FOXO1 (ovarian granulosa cell marker), respectively, indicated that oocytes and follicles were absent in Ddb1fl/fl;Gdf9-Cre ovaries (Fig. 1D and fig. S4, C to E). Complete primordial follicle loss and gonadotropin level increases were observed in these mice within 12 weeks after birth and showed the premature ovarian insufficiency (POI) phenotype (fig. S4, B and F). However, for Ddb1fl/fl;Zp3-Cre mice, DDB1 was intact in oocytes at the primordial follicle stage and was only deleted in activated oocytes. Abundant oocytes remained in Ddb1fl/fl;Zp3-Cre ovaries at 12 weeks (Fig. 1D) to 8 months (fig. S4G) after birth. Thus, DDB1 was essential for oocyte maintenance at the primordial follicle stage.

VPRBP (DCAF1), one of the reported CRL4 adaptors, was also highly expressed in mouse oocytes (figs. S1 and S2). Therefore, we generated oocyte-specific Vprbp knockout mice (Vprbpfl/fl;Gdf9-Cre). VPRBP was deleted in oocytes as early as PD10 (fig. S5A). Vprbpfl/fl;Gdf9-Cre female mice were also infertile (fig. S3B). The ovaries of 12-week-old Vprbpfl/fl;Gdf9-Cre mice were smaller than controls and lost all oocytes (Fig. 1D and fig. S5, B to E). Primordial follicles in Vprbpfl/fl;Gdf9-Cre ovaries were depleted during PD12 to 14 (fig. S5, F and G). Apoptotic oocytes were frequently observed in PD13 Vprbpfl/fl;Gdf9-Cre ovaries (fig. S6, A and B). Oocyte-specific Ddb1 and Vprbp knockout mice were phenocopies of each other, which suggested that these proteins act in the same CRL4 complex to control primordial follicle development.

We postulated that the expression of oocyte-specific genes essential for primordial follicle survival might be altered by Ddb1/Vprbp deletion. Therefore, we determined the mRNA levels of oocyte-enriched genes in Ddb1fl/–;Ddx4-Cre ovaries at PD1. At this time point, the DDB1 protein had already been deleted in oocytes, although normal numbers of oocytes were still found in these ovaries (Fig. 1B and fig. S3C). Many genes essential for oocyte survival, were down-regulated in Ddb1fl/–;Ddx4-Cre ovaries, including Sohlh1/2 (4, 5), Nobox (6), Figla (7), and Kit (8) (Fig. 2A). Decreased expression of essential oocyte genes was also observed in isolated DDB1-deleted oocytes (fig. S4H), as well as in Vprbpfl/fl;Gdf9-Cre ovaries at PD12 (fig. S6C), when VPRBP has been deleted in the oocytes but the primordial follicle numbers have not decreased (fig. S5G). In contrast, the mRNA levels of these genes were comparable to those in control ovaries at PD8 (fig. S6C), a time point before VPRBP deletion in oocytes (fig. S5A). These results indicated that DDB1 or VPRBP deletion resulted in decreased expression of essential genes in oocytes, which contributed to the POI phenotype.

Fig. 2 CRL4VPRBP is required for 5hmC generation and gene expression in primordial follicles.

(A) Quantitative reverse transcription polymerase chain reaction results for the expression of the indicated genes in WT and Ddb1fl/–;Ddx4-Cre ovaries at PD1. The values of different genes in WT were set as “1.” Error bars indicate SEM. (B) Increased DNA methylation in promoters of indicated genes in WT and Ddb1fl/fl;Gdf9-Cre oocytes. Open and filled circles represent unmethylated and methylated CpGs, respectively. Percentages of methylated CpGs are indicated. (C) Immunofluorescence on ovarian sections showing 5hmC levels in primordial follicle stage oocytes (circled by white dots) of WT and Ddb1fl/–;Ddx4-Cre ovaries at PD1. Scale bar, 10 μm. (D) Quantification of oocyte 5hmC signals shown in (C).

We then investigated the mechanism(s) that caused oocyte gene silencing after DDB1 or VPRBP deletion. Because a large numbers of genes were down-regulated in DDB1- or VPRBP-deleted oocytes, we postulated that epigenetic changes in oocytes might have been involved. Indeed, the methylated CpG sites within Gdf9, Figla, Nobox, and Ddx4 promoters were increased in Ddb1fl/fl;Gdf9-Cre oocytes (Fig. 2B and fig. S7A). The paternal imprinting region, H19 DMR, which was slightly methylated in control oocytes, exhibited increased CpG methylation in Ddb1fl/fl;Gdf9-Cre oocytes (fig. S7A). These results indicated that decreased gene expression in Ddb1fl/fl;Gdf9-Cre oocytes might be caused by dysregulated DNA methylation.

To elucidate the mechanism underlying the alteration of gene expression and methylation in Ddb1- or Vprbp-deficient oocytes, we considered the role of TET family DNA dioxygenases, which convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) (9, 10). Notably, 5hmC amounts in DDB1- or VPRBP-deleted oocytes were significantly lower than those in control oocytes at the primordial follicle stage (Fig. 2, C and D, and fig. S7, B and C). TET1, 2, and 3 were highly expressed in oocytes and early embryos (fig. S8A). The decrease in 5hmC generation in DDB1- or VPRBP-deleted oocytes suggested that CRL4VPRBP might regulate TET activities.

Further evidence for the functional relevance of CRL4VPRBP-TET interactions was apparent on the basis of the phenotypes of Ddb1fl/fl;Zp3-Cre female mice. They did not have the POI phenotype but were infertile. Most Ddb1fl/fl;Zp3-Cre oocytes failed to develop beyond the four-cell stage after fertilization (Fig. 3A and fig. S8, B and C). The expression of genes required for early embryo development, including Nanog, Oct4, and Sox2, were not induced in early embryos derived from DDB1-deleted oocytes (fig. S8D). Some other genes, such as Yap and the CRL4 components Cul4b and Roc1, were unchanged. Note that Tet1, 2, and 3 mRNAs were increased in mutant embryos, possibly because of feedback of insufficient TET activity (fig. S8D). Demethylation of markers for successful paternal DNA reprogramming (Line1 and Oct4) was also impaired in DDB1-deleted oocytes (Fig. 3B). These results suggested that DDB1 was a maternal factor required for zygotic genome activation.

Fig. 3 CRL4VPRBP is required for TET3-mediated 5hmC generation and zygotic genome activation after fertilization.

(A) Morphology of blastula derived from Ddb1fl/fl;Zp3-Cre females mated with WT males. Scale bar, 100 μm. (B) Methylation analysis of Line1 and Oct4 promoters in WT and Ddb1fl/fl;Zp3-Cre zygotes. Yellow circles represent unmatched CpGs. Percentages of methylated CpGs are indicated. (C) 5hmC/5mC immunostaining of zygotes from WT and Ddb1fl/fl;Zp3-Cre females 24 hours after mating. PB, polar body. Female and male symbols indicate female and male pronuclei, respectively. Scale bar, 20 μm.

TET3 is involved in epigenetic reprogramming of zygote paternal DNA after natural fertilization and in somatic cell nuclear reprogramming during animal cloning (11). CUL4B, DDB1, and VPRBP were all accumulated in pronuclei after fertilization (fig. S2). 5hmC and TET3 levels were high in the male pronuclei of WT zygotes, but were significantly decreased in zygotes derived from DDB1-deleted oocytes (Fig. 3C and fig. S9A). In somatic cell nuclear transfer experiments, somatic pseudo-pronuclei (PPN) underwent the 5mC-5hmC transition only in reconstructed zygotes derived from WT oocytes but not in those from Ddb1-null oocytes (fig. S9B). TET3 that originated from WT oocytes, but not from DDB1-deleted oocytes, became concentrated in those PPNs (fig. S9C). Thus, the absence of maternal DDB1 blocked zygotic genome reprogramming.

CRL ubiquitin ligase activation requires cullin neddylation, which is catalyzed by NEDD8-activating enzyme E1 (NAE1).To investigate whether CRL4 activity, in addition to the presence of DDB1 and/or VPRBP, was required for 5hmC generation in zygotes, we cultured WT zygotes with MLN4924, an NAE1 inhibitor. MLN4924 abolished cullin neddylation in oocytes (fig. S9D) and 5hmC generation in male pronuclei (fig. S9E). These results indicated that CRL4VPRBP activity was required for TET-mediated 5hmC generation in fertilized oocytes or those with a transferred somatic nucleus.

To determine whether these were general effects, we used HeLa cells as a somatic cell model. DDB1 or VPRBP overexpression increased endogenous 5hmC levels. However, a mutated VPRBP (VPRBP-2RA) that could not bind to DDB1 failed to do so (Fig. 4A and fig. S10A). Overexpression of the TET1 catalytic domain (TET1-CD) induced a significant increase in 5hmC levels. However, this was abolished by DDB1 or VPRBP depletion or with MLN4924 treatment, although TET1 expression and nuclear localization were not affected (Fig. 4B and fig. S10B). By comparison, RNAi depletion of CRL4VPRBP in HeLa cells (fig. S11), as well as MLN4924 treatment, markedly decreased their endogenous 5hmC levels (fig. S10, C to E). These results indicated that CRL4VPRBP was required for TET-mediated 5hmC generation in both oocytes and somatic cells.

Fig. 4 CRL4VPRBP binds to TET enzymes and regulates their activities.

(A) Dot blotting results showing endogenous 5hmC levels after DDB1 or VPRBP overexpression. (B) Dot blotting results showing that TET1 overexpression increased 5hmC levels in HeLa cells; this effect was abolished by both Ddb1 or Vprbp depletion and MLN4924 treatment. (C) Coimmunoprecipitation results showing the interactions between VPRBP and TET1-CD. (D) Impaired DNA binding of TET1/2/3-CD and TET1-FL (full length) after CRL4 inhibition. FLAG tagged TET1/2/3-CD and TET1-FL were expressed and/or purified with or without MLN4924 and were incubated with DNA fragments amplified from Gdf9 and Fgf8 promoters. The TET-bound DNA fragments were detected by PCR and agarose electrophoresis. (E) Illustration of CRL4VPRBP functions in mammalian oocytes. CRL4VPRBP enters the nucleus and activates TETs, which convert 5mC to 5hmC, regulate DNA methylation levels, and maintain the expressions of genes essential for oocyte survival in primordial follicles. When grown oocytes are ovulated and fertilized, CRL4VPRBP recruits TET3 into the male pronucleus and activates zygotic genome reprogramming which is essential for embryo development. WD, WD40.

We next examined if CRL4VPRBP physically interacted with TETs. Coimmunoprecipitation results indicated that TET1, 2, and 3 bound to VPRBP through their C-terminal CD (Fig. 4C and fig. S10F). VPRBP-WT bound to both DDB1 and TET3-CD (fig. S10, G and H). VPRBP-2RA did not bind to DDB1 but still interacted with TET3-CD. Furthermore, the VPRBP N-terminal fragment bound to TET3-CD, although it did not contain the WD40 domain that is essential for DDB1-binding. These results indicated that VPRBP bound to the TET1/2/3-CD through its N-terminal region and bound to DDB1 through its WD40 domain close to its C terminus.

Finally, we investigated the role of CRL4VPRBP in regulating TET1, 2, and 3 activities. TET1/2/3-CD purified from HeLa cells bound with DNA fragments amplified from Gdf9 and Fgf8 promoters or synthesized DNA probes. However, the TETs-DNA interaction was abolished by MLN4924 treatment (Fig. 4D and fig. S10I). Taken together, we established that CRL4VPRBP was essential for TET1, 2, and 3 activities by promoting their DNA binding ability.

In recent years, phosphatidylinositol 3-kinase signaling in oocytes has attracted attention as a determinant of ovarian aging (1214). However, whether other pathways in oocytes also have direct effects on maintaining the primordial follicle pool remains uncertain. Our results identified the E3 ligase CRL4VPRBP as a crucial factor for oocyte survival. Rather than activating primordial follicles, CRL4VPRBP regulates their survival or loss to determine reproductive aging and menopause in females. The mouse models generated in this study (summarized in table S1) mimicked human POI patients better than previously reported Foxo3a- or Pten-deleted mice in several respects. For example, Foxo3a or Pten deletion in mouse oocytes resulted in (i) global activation of an ovarian primordial follicle pool; (ii) increased ovulation of activated oocytes; and, ultimately, (iii) female germ cell exhaustion (12, 14). However, the first two steps are not observed in most human POI patients. Typically, even if oocytes are eventually obtained from these patients, they fail to develop into healthy embryos after in vitro fertilization. These phenomena cannot be simply explained by rapid oocyte exhaustion. Our study with oocyte-specific CRL4VPRBP-deficient mice provides new evidence that in genetically defective females that are destined for POI, mutated but otherwise “healthy-looking” oocytes could be epigenetically abnormal and have limited developmental potential, even before their physical disappearance. The role of CRL4VPRBP in mammalian oocytes is summarized in Fig. 4E.

Although CRL4VPRBP is crucial for TET activities, our results suggest that TET1, 2, and 3 are not the only CRL4VPRBP substrates in oocytes. More than one-third of the embryos derived from TET3-deleted oocytes could develop to term (11). However, all embryos that were derived from DDB1-deleted oocytes died before the eight-cell stage, which indicated that they had defects other than TET3-mediated genome reprogramming. CRL4 might also recruit other substrate adaptors, poly-ubiquitinate a number of protein substrates, and direct them toward degradation. Identifying other CRL4VPRBP substrates will shed new light on the molecular regulatory mechanisms of oocyte functions.

Supplementary Materials

www.sciencemag.org/content/342/6165/1518/suppl/DC1

Materials and Methods

Figs. S1 to S11

Tables S1 to S3

References (1524)

References and Notes

  1. Acknowledgments: We thank K. Guan and X. Feng for discussions and critical reviews of the manuscript. J.J. Chen and Z. Xia provided VPRBP and TET1, 2, and 3 plasmids, respectively. G. Xu and L. Jia provided TET3 antibody and MLN4924, respectively. Y. Xiong provided Vprbp floxed mice and VPRBP-2RA plasmid. The authors wish to acknowledge contributions of Tadashi Nakagawa and Yue Xiong (University of North Carolina—Chapel Hill). Nakagawa and Xiong initiated the VprBP-Tet project and made significant contributions to the published study. Xiong's lab generated and provided us the VprBP conditional mutant mice on the basis of direct collaboration in summer 2012. Nakagawa, a postdoctoral fellow in Xiong's lab, discovered the VprBP-Tet interaction by immunoprecipitation and mass spectrometry analysis of the VprBP immunocomplex in early 2012. Nakagawa also discovered in 2012, through knocking down and knockout VprBP in cultured human cells and in mouse embryo fibroblasts, that the function of VprBP is critically important for the activity of Tet. Xiong has communicated these and other unpublished results to us in multiple communications between August 2012 and April 2013. This study was supported by National Basic Research Program of China [2011CB944504 and 2012CB944403 (to H.-Y.F. and Q.-Y.S.) and 2012CB966600 (to C.T.)], National Natural Science Foundation of China (81172473 and 31371449 to H.-Y.F.), and Basic Scientific Research Funding of Zhejiang University (2011QN81001 to H.-Y.F).
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