CELL CYCLE REGULATION DURING OOGENESIS
, Head, Unit on Cell Cycle Regulation
Eve Feinberg, MD, Clinical Fellow
Eva Decotto, PhD, Visiting Fellow
Karine Narbonne, PhD, Visiting Fellow
Stefania Senger, PhD, Visiting Fellow
P27/Dacapo and the licensing of DNA replication origins in Drosophila
The endocycle is a developmentally programmed variant cell cycle in which cells undergo repeated rounds of DNA replication with no intervening mitosis. In Drosophila, the oscillations of Cyclin E/Cdk2 activity drive the endocycle. How the periodicity of Cyclin E/Cdk2 activity is achieved during endocycles is poorly understood. We have determined that the p21cip/p27kip1/p57kip2-like Cyclin-dependent kinase inhibitor (CKI) Dacapo (Dap) promotes replication licensing during Drosophila endocycles by reinforcing low Cdk activity during the endocycle Gap phase. In dap mutants, cells in the endocycle exhibit reduced levels of the licensing factor Double-Parked/Cdt1 (Dup/Cdt1) as well as decreased levels of chromatin-bound MCM2-7 complex. In addition, mutations in dup/cdt1 dominantly enhance the dap phenotype in several polyploid cell types. Consistent with their reduced ability to complete genomic replication, dap mutants accumulate increased levels of DNA damage during the endocycle S phase. Intriguingly, we find that dap also promotes replication licensing and genomic stability during pre-meiotic S phase. Our data suggest a model in which Dap inhibits Cyclin E/Cdk2 activity during the Gap phase and thus promotes the efficient licensing of DNA replication origins. A similar role has been defined for the CKI SIC1 in promoting replication origin licensing in late G1 in S. cerevisiae. However, our work represents the first report of a CKI acting to promote replication licensing in a metazoan.
Hong A, Narbonne-Reveau K, Riesgo-Escovar J, Fu H, Aladjem MI, Lilly MA. The cyclin-dependent kinase inhibitor Dacapo promotes replication licensing during Drosophila endocycles. EMBO J 2007;26:2071-82.
Translational control of mitotic Cyclin expression in prophase I–arrested oocytes
Arrest of the oocyte cell cycle in prophase of meiosis I (prophase I) is a universally conserved feature of animal oogenesis. During prophase I arrest, animal oocytes must perform two seemingly contradictory tasks. First, they must accumulate and store large quantities of mRNAs and proteins that are required to drive the two future meiotic divisions as well as the mitotic divisions of the early embryo. Second, they must remain fully arrested in order to maintain the integrity of the genome and avoid producing an aneuploid gamete. While arrest of the oocyte cell cycle in prophase I is one of the most highly conserved events of gametogenesis, the precise developmental and cell cycle events that initiate and maintain the arrest are not fully understood.
We have determined that the translational inhibitor Bruno, which is encoded by the arrest gene, maintains mitotic quiescence during the prophase I meiotic arrest of the Drosophila oocyte. In arrest(bruno) mutants, ovarian cysts enter the meiotic cycle and progress to pachytene, as indicated by the formation of mature synaptonemal complexes. However, after meiotic entry, the levels of the mitotic Cyclins increase, and the germ cells re-enter the mitotic cycle and continue to proliferate. Thus, Bruno functions to inhibit the expression of the mitotic Cyclins after meiotic entry. Our data indicate that Bruno accomplishes its task in part by binding to Bruno Response Elements (BREs) present in the cyclin A 3′UTR and inhibiting its translation. In Drosophila, Cyclin A is the primary positive regulatory subunit of Cdk1. Clams and fish, as well as many amphibians, employ a similar strategy for maintaining both the prophase I arrest and the required low levels of Cdk1 activity, whereby the translation of Cyclin B is inhibited until meiotic maturation. Bruno has previously been implicated in the translational inhibition of gurken and oskar, two genes involved in the differentiation of the egg and embryo. The dual function of Bruno in regulating the translation of genes that influence both the meiotic program and oocyte differentiation suggests a model for how cell cycle regulation and gamete differentiation are coordinated during oogenesis. Our findings represent a major step forward in understanding the regulation of the early meiotic cycle in a genetically tractable metazoan and will provide a framework for future studies on the regulation of highly conserved cell cycle arrest.
To define further the developmental inputs that control meiotic progression during oogenesis, we have initiated genetic screens to identify the pathways that regulate Bruno expression and activity during the prophase I meiotic arrest.
Mavrakis M, Rikhy R, Lilly MA, Lippincott-Schwartz J. Fluorescence imaging techniques for studying Drosophila development. Curr Protocols Cell Biol 2007, in press.
Sugimura I, Lilly MA. Bruno restricts the accumulation of the mitotic Cyclins during the prophase I meiotic arrest of the Drosophila oocyte. Dev Cell 2006;10:127-35.
Coordinating meiotic progression and oocyte differentiation
A long-term goal of our laboratory has been to identify factors that are concentrated or activated in the oocyte and that promote meiotic progression and/or establishment of oocyte identity. To identify the pathways that direct entry into and maintenance of the meiotic cycle, we screened for mutants in which ovarian cysts develop with 16 nurse cells and no oocyte. From this screen, we identified missing oocyte (mio), a new gene that is required for the maintenance of the meiotic cycle. In mio mutants, the oocyte enters the meiotic cycle and forms mature synaptonemal complexes but does not maintain the meiotic state. Ultimately, mio oocytes abandon the meiotic cycle, enter the endocycle, and develop as nurse cells. Intriguingly, inhibition of the formation of the double-stranded breaks that initiate meiotic recombination strongly suppresses the mio 16–nurse-cell phenotype. The data suggest that mio interacts with pathways that influence DNA metabolism. mio is predicted to encode a protein of 867 amino acids that is highly conserved from yeast to humans. The conservation of the Mio protein is present in two main blocks. First, the N-terminal block contains four to six WD-40 repeats, which often function as protein-protein interaction domains. Second, the C-terminal block of the protein contains a putative U box, which is structurally similar to the RING finger domain and has been implicated in ubiquitin-dependent protein degradation. We initiated a biochemical characterization of Mio. From these studies, we found that Mio is present in a stable multiprotein complex of approximately 550 kDa. In addition, we determined that Mio physically and genetically interacts with components of the nuclear pore.
1 Isamu Sugimura, PhD, former Visiting Fellow
COLLABORATORS
Mirit Aladjem, PhD, Laboratory of Molecular Pharmacology, NCI, Bethesda, MD
Juan Riesgo-Escovar, PhD, Neurobiology Institute, Campus UNAM-Juriquilla, Querétaro, Mexico
For further information, contact mary_lilly@nih.gov.
