CYLD deficiency enhances metabolic reprogramming and tumor progression in nasopharyngeal carcinoma via PFKFB3.

Aberrant cancer metabolism contributes to cell proliferation and tumor progression. However, the contribution of enhanced glycolysis, observed during cancer metabolism, to the pathogenesis and progression of nasopharyngeal carcinoma (NPC) remains unclear. CYLD, an NF-κB inhibitor, is frequently deficient in NPC. Here, we investigated the role of CYLD in the metabolic reprogramming of NPC and found that restoration of CYLD expression suppressed glycolysis in NPC cells. Mechanistic dissection showed that CYLD stabilized p53 and facilitated its nuclear translocation, thereby enhancing p53 activity by removing K63-linked and K48-linked ubiquitin chains of p53, which can bind to the PFKFB3 promoter and inhibit its transcription. Additionally, CYLD interacted with FZR1 to promote APC/C-FZR1 E3 ligase activity, which further ubiquitinated and degraded PFKFB3 via the 26S proteasomal system. Furthermore, clinical tissue array analysis indicated that low expression of CYLD was correlated with high expression of PFKFB3 and poor prognosis among patients with NPC. In conclusion, CYLD suppressed PFKFB3 expression via two factors, namely, p53 and FZR1, to inhibit glycolysis and delay tumor growth and progression in NPC. CYLD is a biomarker indicating poor prognosis of patients with NPC.

De novo FZR1 loss-of-function variants cause developmental and epileptic encephalopathies.

FZR1, which encodes the Cdh1 subunit of the anaphase-promoting complex, plays an important role in neurodevelopment by regulating the cell cycle and by its multiple post-mitotic functions in neurons. In this study, evaluation of 250 unrelated patients with developmental and epileptic encephalopathies and a connection on GeneMatcher led to the identification of three de novo missense variants in FZR1. Whole-exome sequencing in 39 patient-parent trios and subsequent targeted sequencing in an additional cohort of 211 patients was performed to identify novel genes involved in developmental and epileptic encephalopathy. Functional studies in Drosophila were performed using three different mutant alleles of the Drosophila homologue of FZR1 fzr. All three individuals carrying de novo variants in FZR1 had childhood-onset generalized epilepsy, intellectual disability, mild ataxia and normal head circumference. Two individuals were diagnosed with the developmental and epileptic encephalopathy subtype myoclonic atonic epilepsy. We provide genetic-association testing using two independent statistical tests to support FZR1 association with developmental and epileptic encephalopathies. Further, we provide functional evidence that the missense variants are loss-of-function alleles using Drosophila neurodevelopment assays. Using three fly mutant alleles of the Drosophila homologue fzr and overexpression studies, we show that patient variants can affect proper neurodevelopment. With the recent report of a patient with neonatal-onset with microcephaly who also carries a de novo FZR1 missense variant, our study consolidates the relationship between FZR1 and developmental and epileptic encephalopathy and expands the associated phenotype. We conclude that heterozygous loss-of-function of FZR1 leads to developmental and epileptic encephalopathies associated with a spectrum of neonatal to childhood-onset seizure types, developmental delay and mild ataxia. Microcephaly can be present but is not an essential feature of FZR1-encephalopathy. In summary, our approach of targeted sequencing using novel gene candidates and functional testing in Drosophila will help solve undiagnosed myoclonic atonic epilepsy or developmental and epileptic encephalopathy cases.

FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

AIMS: Vascular smooth muscle cells (VSMCs) normally exhibit a very low proliferative rate. Vessel injury triggers VSMC proliferation, in part, through focal adhesion kinase (FAK) activation, which increases transcription of cyclin D1, a key activator for cell cycle-dependent kinases (CDKs). At the same time, we also observe that FAK regulates the expression of the CDK inhibitors (CDKIs) p27 and p21. However, the mechanism of how FAK controls CDKIs in cell cycle progression is not fully understood. METHODS AND RESULTS: We found that pharmacological and genetic FAK inhibition increased p27 and p21 by reducing stability of S-phase kinase-associated protein 2 (Skp2), which targets theCDKIs for degradation. FAK N-terminal domain interacts with Skp2 and an APC/C E3 ligase activator fizzy-related 1 (Fzr1) in the nucleus, which promote ubiquitination and degradation of both Skp2 and Fzr1. Notably, overexpression of cyclin D1 alone failed to promote proliferation of genetic FAK kinase-dead (KD) VSMCs, suggesting that the FAK-Skp2-CDKI signalling axis is distinct from the FAK-cyclin D1 pathway. However, overexpression of both cyclin D1 and Skp2 enabled proliferation of FAK-KD VSMCs, implicating that FAK ought to control both activating and inhibitory switches for CDKs. In vivo, wire injury activated FAK in the cytosol, which increased Skp2 and decreased p27 and p21 levels. CONCLUSION: Both pharmacological FAK and genetic FAK inhibition reduced Skp2 expression in VSMCs upon injury, which significantly reduced intimal hyperplasia through elevated expression of p27 and p21. This study revealed that nuclear FAK-Skp2-CDKI signalling negatively regulates CDK activity in VSMC proliferation.

RNA methyltransferase NSUN2 promotes growth of hepatocellular carcinoma cells by regulating fizzy-related-1 in vitro and in vivo.

The aim of the study was to investigate the role of NSUN2 (NOP2/Sun RNA Methyltransferase Family Member 2) in hepatocellular carcinoma (HCC). The expressions of NSUN2 and FZR1 were measured. Cell viability, proliferation, and apoptosis were assessed. HCC xenograft in nude mouse model was established. Tumor weight and volume were examined. Tumor tissues were collected for immunohistochemistry (IHC). TCGA database analysis and clinical sample testing suggested that the transcript levels of NSUN2 and FZR1 were increased in HCC tissues. NSUN2 knockdown inhibited HCC cell viability and proliferation, and promoted cell apoptosis. Moreover, the effects of NSUN2 could be countered by overexpressing FZR1. In animal experiment, NSUN2 silencing suppressed tumor growth in nude mice by downregulating FZR1. In conclusion, NSUN2 has a regulatory effect on HCC cell proliferation and apoptosis. NSUN2 knockout could inhibit cellular processes in HCC and tumor growth, likely via FZR1 inhibition. This finding has not only revealed the role of NSUN2 in HCC growth, but also suggests a promising target for HCC treatment.

A Novel Signature of CCNF-Associated E3 Ligases Collaborate and Counter Each Other in Breast Cancer.

Breast cancer (BRCA) malignancy causes major fatalities amongst women worldwide. SCF (Skp1-cullin-F-box proteins) E3 ubiquitin ligases are the most well-known members of the ubiquitination-proteasome system (UPS), which promotes cancer initiation and progression. Recently, we demonstrated that FBXL8, a novel F-box protein (SCFF-boxes) of SCF E3 ligase, accelerates BRCA advancement and metastasis. Since SCFF-boxes is a key component of E3 ligases, we hypothesized that other SCFF-boxes besides FBXL8 probably collaborate in regulating breast carcinogenesis. In this study, we retrospectively profiled the transcriptome of BRCA tissues and found a notable upregulation of four SCFF-box E3 ligases (FBXL8, FBXO43, FBXO15, and CCNF) in the carcinoma tissues. Similar to FBXL8, the knockdown of FBXO43 reduced cancer cell viability and proliferation, suggesting its pro-tumorigenic role. The overexpression of CCNF inhibited cancer cell progression, indicating its anti-tumorigenic role. Unexpectedly, CCNF protein was markedly downregulated in BRCA tissues, although its mRNA level was high. We showed that both E3 ligases, FBXL8 and FZR1, pulled down CCNF. Double knockdown of FBXL8 and FZR1 caused CCNF accumulation. On the other hand, CCNF itself pulled down a tumorigenic factor, RRM2, and CCNF overexpression reduced RRM2. Altogether, we propose a signature network of E3 ligases that collaboratively modulates CCNF anti-cancer activity. There is potential to target BRCA through modulation of the partnership axes of (i) CCNF-FBXL8, (ii) CCNF-FZR1, and (iii) CCNF-RRM2, particularly, via CCNF overexpression and activation and FBXL8/FZR1 suppression.

APC/CFZR-1 regulates centrosomal ZYG-1 to limit centrosome number.

Aberrant centrosome numbers are associated with human cancers. The levels of centrosome regulators positively correlate with centrosome number. Thus, tight control of centrosome protein levels is critical. In Caenorhabditis elegans, the anaphase-promoting complex/cyclosome and its co-activator FZR-1 (APC/CFZR-1), a ubiquitin ligase, negatively regulates centrosome assembly through SAS-5 degradation. In this study, we report the C. elegans ZYG-1 (Plk4 in humans) as a potential substrate of APC/CFZR-1. Inhibiting APC/CFZR-1 or mutating a ZYG-1 destruction (D)-box leads to elevated ZYG-1 levels at centrosomes, restoring bipolar spindles and embryonic viability to zyg-1 mutants, suggesting that APC/CFZR-1 influences centrosomal ZYG-1 via the D-box motif. We also show the Slimb/ßTrCP-binding (SB) motif is critical for ZYG-1 degradation, substantiating a conserved mechanism by which ZYG-1/Plk4 stability is regulated by the SKP1-CUL1-F-box (Slimb/ßTrCP)-protein complex (SCFSlimb/ßTrCP)-dependent proteolysis via the conserved SB motif in C. elegans. Furthermore, we show that co-mutating ZYG-1 SB and D-box motifs stabilizes ZYG-1 in an additive manner, suggesting that the APC/CFZR-1 and SCFSlimb/ßTrCP ubiquitin ligases function cooperatively for timely ZYG-1 destruction in C. elegans embryos where ZYG-1 activity remains at threshold level to ensure normal centrosome number.

LxCxD motif of the APC/C coactivator subunit FZR1 is critical for interaction with the retinoblastoma protein.

Retinoblastoma protein (pRB) regulates cell cycle by utilizing different regions of its pocket domain for interacting with E2F family of transcription factors and with cellular and viral proteins containing an LxCxE motif. An LxCxE-like motif, LxCxD, is present in FZR1, an adaptor protein of the multi-subunit E3 ligase complex anaphase-promoting complex/cyclosome (APC/C). The APC/CFZR1 complex regulates the timely degradation of multiple cell cycle proteins for mitotic exit and maintains G1 state. We report that FZR1 interacts with pRB via its LxCxD motif. By using point mutations, we found that the cysteine residue in the FZR1 LxCxD motif is critical for direct interaction with pRb. The direct binding of the LxCxD motif of FZR1 to the pRB LxCxE binding pocket is confirmed by using human papillomavirus protein E7 as a competitor, both in vitro and in vivo. While mutation of the cysteine residue significantly disrupts FZR1 interaction with pRB, this motif does not affect FZR1 and core APC/C association. Expression of the FZR1 point mutant results in accumulation of S-phase kinase-associated protein 2 (SKP2) and Polo-like kinase 1 (PLK1), while p27Kip1 and p21Cip1 proteins are downregulated, indicating a G1 cell cycle defect. Consistently, cells containing point mutant FZR1 enter the S phase prematurely. Together our results suggest that the LxCxD motif of FZR1 is a critical determinant for the interaction between FZR1 and pRB and is important for G1 restriction.

Phosphorylation regulates cullin-based ubiquitination in tumorigenesis.

Cullin-RING ligases (CRLs) recognize and interact with substrates for ubiquitination and degradation, and can be targeted for disease treatment when the abnormal expression of substrates involves pathologic processes. Phosphorylation, either of substrates or receptors of CRLs, can alter their interaction. Phosphorylation-dependent ubiquitination and proteasome degradation influence various cellular processes and can contribute to the occurrence of various diseases, most often tumorigenesis. These processes have the potential to be used for tumor intervention through the regulation of the activities of related kinases, along with the regulation of the stability of specific oncoproteins and tumor suppressors. This review describes the mechanisms and biological functions of crosstalk between phosphorylation and ubiquitination, and most importantly its influence on tumorigenesis, to provide new directions and strategies for tumor therapy.

CDH1 binds MAD2L2 in a Rev1-like pattern.

MAD2L2 (i.e. Rev7) is a central regulatory protein important in several processes, such as translesion synthesis (TLS), DNA damage response and mitosis. In TLS, MAD2L2 binds Rev3 to form Pol zeta (ζ) and promotes formation of the Pol ζ- REV1 complex allowing extension beyond distorted DNA structures. MAD2L2 is also part of the heterotetrameric shieldin complex that regulates DNA repair at sites of damage, where similarly to TLS, it bridges the interaction between SHLD2 and SHLD3. Lastly, during mitosis, MAD2L2 prevents premature activation of the anaphase promoting complex/cyclosome (APC/C), by sequestering its activator, CDH1. MAD2L2 exits in a 'closed' active conformation binding Rev3 and Rev1, or SHLD2 and SHLD3, and an 'open' inactive conformation, with no binding partners. Moreover, Pol ζ- REV1 forms a homodimer using a protein-protein interaction (PPI) domain comprised of a central αC helix, promoting Rev3-MAD2L2 interaction and C-terminus ß-sheets, enabling Rev1-MAD2L2 interaction. While the role of MAD2L2 in TLS is well established, molecular details regarding the CDH1-MAD2L2 interaction and MAD2L2 homodimerization are still missing. Here we demonstrate, in a human cell line, using a series of MAD2L2 mutants, that MAD2L2's C-terminus interface is essential for the CDH1-MAD2L2 binding as well as for homodimerization. In addition, we show that CDH1 interacts with MAD2L2 in a Rev1-like pattern, using the same C-terminus residues on MAD2L2 which Rev1 binds. Thus, identification of CDH1 as an additional Rev1-like binding protein strengthens the versatility of MAD2L2 as a regulatory protein and emphasizes the complexity involved in MAD2L2's preferential complex formation.

AURKA destruction is decoupled from its activity at mitotic exit but is essential to suppress interphase activity.

Activity of AURKA is controlled through multiple mechanisms including phosphorylation, ubiquitin-mediated degradation and allosteric interaction with TPX2. Activity peaks at mitosis, before AURKA is degraded during and after mitotic exit in a process strictly dependent on the APC/C coactivator FZR1. We used FZR1 knockout cells (FZR1KO) and a novel FRET-based AURKA biosensor to investigate how AURKA activity is regulated in the absence of destruction. We found that AURKA activity in FZR1KO cells dropped at mitotic exit as rapidly as in parental cells, despite absence of AURKA destruction. Unexpectedly, TPX2 was degraded normally in FZR1KO cells. Overexpression of an N-terminal TPX2 fragment sufficient for AURKA binding, but that is not degraded at mitotic exit, caused delay in AURKA inactivation. We conclude that inactivation of AURKA at mitotic exit is determined not by AURKA degradation but by degradation of TPX2 and therefore is dependent on CDC20 rather than FZR1. The biosensor revealed that FZR1 instead suppresses AURKA activity in interphase and is critically required for assembly of the interphase mitochondrial network after mitosis.This article has an associated First Person interview with the first authors of the paper.

CENP-T regulates both the G2/M transition and anaphase entry by acting through CDH1 in meiotic oocytes.

Oocyte meiotic maturation failure is one of the major causes for female infertility. Meiotic resumption (the G2/M transition) and progression through metaphase I (MI) are two critical stages of oocyte meiotic maturation. Here, we report that centromere protein T (CENP-T), an internal kinetochore protein, plays a critical role in meiotic resumption of mouse oocytes. Depletion of CENP-T by siRNA injection increased the CDH1 (also known as FZR1) level, resulting in increased activity of the anaphase-promoting complex (APC)-CDH1 complex, and further leading to decreased levels of the cyclin protein CCNB1, attenuated maturation-promoting factor (MPF) activity, and finally severely compromised meiotic resumption. The impaired meiotic resumption caused by CENP-T depletion could be rescued by overexpression of exogenous CCNB1 or knockdown of endogenous CDH1. Overexpression of exogenous CENP-T resulted in decreased CDH1 levels, which accelerated the progression of G2/M transition, and accelerated meiotic cell cycle progression after germinal vesicle breakdown (GVBD). Unexpectedly, spindle organization after GVBD was not affected by the overexpression, but the distribution of chromosomes was affected. Our findings reveal a novel role for CENP-T in regulating meiotic progression by acting through CDH1.

A novel human Cdh1 mutation impairs anaphase promoting complex/cyclosome activity resulting in microcephaly, psychomotor retardation, and epilepsy.

The Fizzy-related protein 1 (Fzr1) gene encodes Cdh1 protein, a coactivator of the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). Previously, we found that genetic ablation of Fzr1 promotes the death of neural progenitor cells leading to neurogenesis impairment and microcephaly in mouse. To ascertain the possible translation of these findings in humans, we searched for mutations in the Fzr1 gene in 390 whole exomes sequenced in trio in individuals showing neurodevelopmental disorders compatible with a genetic origin. We found a novel missense (p.Asp187Gly) Fzr1 gene mutation (c.560A>G) in a heterozygous state in a 4-year-old boy, born from non-consanguineous Spanish parents, who presents with severe antenatal microcephaly, psychomotor retardation, and refractory epilepsy. Cdh1 protein levels in leucocytes isolated from the patient were significantly lower than those found in his parents. Expression of the Asp187Gly mutant form of Cdh1 in human embryonic kidney 293T cells produced less Cdh1 protein and APC/C activity, resulting in altered cell cycle distribution when compared with cells expressing wild-type Cdh1. Furthermore, ectopic expression of the Asp187Gly mutant form of Cdh1 in cortical progenitor cells in primary culture failed to abolish the enlargement of the replicative phase caused by knockout of endogenous Cdh1. These results indicate that the loss of function of APC/C-Cdh1 caused by Cdh1 Asp187Gly mutation is a new cause of prenatal microcephaly, psychomotor retardation, and severe epilepsy. Read the Editorial Highlight for this article on page 8. Cover Image for this issue: doi: 10.1111/jnc.14524.

APC/CFZR-1 Controls SAS-5 Levels To Regulate Centrosome Duplication in Caenorhabditis elegans.

As the primary microtubule-organizing center, centrosomes play a key role in establishing mitotic bipolar spindles that secure correct transmission of genomic content. For the fidelity of cell division, centrosome number must be strictly controlled by duplicating only once per cell cycle. Proper levels of centrosome proteins are shown to be critical for normal centrosome number and function. Overexpressing core centrosome factors leads to extra centrosomes, while depleting these factors results in centrosome duplication failure. In this regard, protein turnover by the ubiquitin-proteasome system provides a vital mechanism for the regulation of centrosome protein levels. Here, we report that FZR-1, the Caenorhabditis elegans homolog of Cdh1/Hct1/Fzr, a coactivator of the anaphase promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase, functions as a negative regulator of centrosome duplication in the C. elegans embryo. During mitotic cell division in the early embryo, FZR-1 is associated with centrosomes and enriched at nuclei. Loss of fzr-1 function restores centrosome duplication and embryonic viability to the hypomorphic zyg-1(it25) mutant, in part, through elevated levels of SAS-5 at centrosomes. Our data suggest that the APC/CFZR-1 regulates SAS-5 levels by directly recognizing the conserved KEN-box motif, contributing to proper centrosome duplication. Together, our work shows that FZR-1 plays a conserved role in regulating centrosome duplication in C. elegans.

Fission yeast APC/C activators Slp1 and Fzr1 sequentially trigger two consecutive nuclear divisions during meiosis.

In meiosis, two rounds of nuclear division occur consecutively without DNA replication between the divisions. We isolated a fission yeast mutant in which the nucleus divides only once to generate two spores, as opposed to four, in meiosis. In this mutant, we found that the initiation codon of the slp1+ gene is converted to ATA, producing a reduced amount of Slp1. As a member of the Fizzy family of anaphase-promoting complex/cyclosome (APC/C) activators, Slp1 is essential for vegetative growth; however, the mutant allele shows a phenotype only in meiosis. Slp1 insufficiency delays degradation of maturation-promoting factor at the first meiotic division, and another APC/C activator, Fzr1, which acts late in meiosis, terminates meiosis immediately after the delayed first division to produce two viable spores.

CenpH regulates meiotic G2/M transition by modulating the APC/CCdh1-cyclin B1 pathway in oocytes.

Meiotic resumption (G2/M transition) and progression through meiosis I (MI) are two key stages for producing fertilization-competent eggs. Here, we report that CenpH, a component of the kinetochore inner plate, is responsible for G2/M transition in meiotic mouse oocytes. Depletion of CenpH by morpholino injection decreased cyclin B1 levels, resulting in attenuation of maturation-promoting factor (MPF) activation, and severely compromised meiotic resumption. CenpH protects cyclin B1 from destruction by competing with the action of APC/CCdh1 Impaired G2/M transition after CenpH depletion could be rescued by expression of exogenous cyclin B1. Unexpectedly, blocking CenpH did not affect spindle organization and meiotic cell cycle progression after germinal vesicle breakdown. Our findings reveal a novel role of CenpH in regulating meiotic G2/M transition by acting via the APC/CCdh1-cyclin B1 pathway.

Identification of the APC/C co-factor FZR1 as a novel therapeutic target for multiple myeloma.

Multiple Myeloma (MM) is a haematological neoplasm characterised by the clonal proliferation of malignant plasma cells in the bone marrow. The success of proteasome inhibitors in the treatment of MM has highlighted the importance of the ubiquitin proteasome system (UPS) in the pathogenesis of this disease. In this study, we analysed gene expression of UPS components to identify novel therapeutic targets within this pathway in MM. Here we demonstrate how this approach identified previously validated and novel therapeutic targets. In addition we show that FZR1 (Fzr), a cofactor of the multi-subunit E3 ligase complex anaphase-promoting complex/cyclosome (APC/C), represents a novel therapeutic target in myeloma. The APC/C associates independently with two cofactors, Fzr and Cdc20, to control cell cycle progression. We found high levels of FZR1 in MM primary cells and cell lines and demonstrate that expression is further increased on adhesion to bone marrow stromal cells (BMSCs). Specific knockdown of either FZR1 or CDC20 reduced viability and induced growth arrest of MM cell lines, and resulted in accumulation of APC/CFzr substrate Topoisomerase IIα (TOPIIα) or APC/CCdc20 substrate Cyclin B. Similar effects were observed following treatment with proTAME, an inhibitor of both APC/CFzr and APC/CCdc20. Combinations of proTAME with topoisomerase inhibitors, etoposide and doxorubicin, significantly increased cell death in MM cell lines and primary cells, particularly if TOPIIα levels were first increased through pre-treatment with proTAME. Similarly, combinations of proTAME with the microtubule inhibitor vincristine resulted in enhanced cell death. This study demonstrates the potential of targeting the APC/C and its cofactors as a therapeutic approach in MM.

APC/C and retinoblastoma interaction: cross-talk of retinoblastoma protein with the ubiquitin proteasome pathway.

The ubiquitin (Ub) ligase anaphase promoting complex/cyclosome (APC/C) and the tumour suppressor retinoblastoma protein (pRB) play key roles in cell cycle regulation. APC/C is a critical regulator of mitosis and G1-phase of the cell cycle whereas pRB keeps a check on proliferation by inhibiting transition to the S-phase. APC/C and pRB interact with each other via the co-activator of APC/C, FZR1, providing an alternative pathway of regulation of G1 to S transition by pRB using a post-translational mechanism. Both pRB and FZR1 have complex roles and are implicated not only in regulation of cell proliferation but also in differentiation, quiescence, apoptosis, maintenance of chromosomal integrity and metabolism. Both are also targeted by transforming viruses. We discuss recent advances in our understanding of the involvement of APC/C and pRB in cell cycle based decisions and how these insights will be useful for development of anti-cancer and anti-viral drugs.

The C. elegans NR4A nuclear receptor gene nhr-6 promotes cell cycle progression in the spermatheca lineage.

BACKGROUND: NR4A nuclear receptors are a conserved, functionally diverse group of nuclear receptors that regulate multiple cellular processes including proliferation and differentiation. The gene nhr-6 encodes the sole Caenorhabditis elegans NR4A nuclear receptor homolog with an essential role in reproduction by regulating morphogenesis of the spermatheca, a somatic gonad organ involved in ovulation and fertilization. RESULTS: Here, we identify the spermatheca cell lineage defects that occur in nhr-6 mutants. Utilizing cell marker analysis, we find that nhr-6 is required for cell cycle progression and that the cell proliferation phenotype is not due to premature cell cycle exit. We also show that loss of the negative cell cycle regulators fzr-1 and lin-35 suppresses the cell proliferation defects. We further demonstrate that NHR-6 activity intersects with Eph receptor signaling during spermatheca cell proliferation. CONCLUSIONS: NHR-6 has an essential function in promoting cell cycle progression during G1 phase in a specific spermatheca cell lineage. Genetic suppression of the proliferation phenotype does not affect the differentiation phenotypes observed in nhr-6 mutants, indicating a dualistic role for nhr-6 in regulating cell proliferation and cell differentiation during spermatheca organogenesis.

The APC/C activator FZR1 is essential for meiotic prophase I in mice.

Fizzy-related 1 (FZR1) is an activator of the Anaphase promoting complex/cyclosome (APC/C) and an important regulator of the mitotic cell division cycle. Using a germ-cell-specific conditional knockout model we examined its role in entry into meiosis and early meiotic events in both sexes. Loss of APC/C(FZR1) activity in the male germline led to both a mitotic and a meiotic testicular defect resulting in infertility due to the absence of mature spermatozoa. Spermatogonia in the prepubertal testes of such mice had abnormal proliferation and delayed entry into meiosis. Although early recombination events were initiated, male germ cells failed to progress beyond zygotene and underwent apoptosis. Loss of APC/C(FZR1) activity was associated with raised cyclin B1 levels, suggesting that CDK1 may trigger apoptosis. By contrast, female FZR1Δ mice were subfertile, with premature onset of ovarian failure by 5 months of age. Germ cell loss occurred embryonically in the ovary, around the time of the zygotene-pachytene transition, similar to that observed in males. In addition, the transition of primordial follicles into the growing follicle pool in the neonatal ovary was abnormal, such that the primordial follicles were prematurely depleted. We conclude that APC/C(FZR1) is an essential regulator of spermatogonial proliferation and early meiotic prophase I in both male and female germ cells and is therefore important in establishing the reproductive health of adult male and female mammals.