Overview

The regulation of metazoan DNA replication and chromosome segregation. The coordination of cell division and the cell cycle with development.

Our research goal is to decipher how the two fundamental steps in cell division, DNA replication and chromosome segregation, are regulated during the development of multicellular organisms. This coordination not only involves controlling the number of cell division cycles, but also the implementation of modified cell cycles for particular developmental strategies. Haploid gametes are produced by meiosis, and in oogenesis the meiotic cell cycle is linked to oocyte differentiation by developmentally triggered arrest and release points. Organisms that undergo rapid embryogenesis utilize an abbreviated cell cycle without growth phases. A third variant cell cycle, the endo cycle, is employed in specific tissues in most plants and animals. In the endo cycle DNA replication occurs but not mitosis, producing large polyploid or polytene cells with high metabolic activity. To investigate the interface between cell cycle and development we are using the fruit fly, Drosophila melanogaster, because variant cell cycles are employed throughout its development. In addition to these intriguing aspects of cell cycle regulation, Drosophila has technical advantages that facilitate analysis of DNA replication and chromosome segregation. Mutants can be recovered and their defects precisely defined, in large part because replication origins, chromosomal proteins, and chromosome segregation can be visualized directly. These attributes make Drosophila ideal for gene discovery, and we have identified proteins crucial for chromosome segregation and DNA replication that are conserved to humans.

Research Summary

Developmental regulation of meiosis: During oogenesis progression through meiosis must be coordinated with developmental events. In most organisms oocytes arrest in meiosis I prophase to permit oocyte differentiation and at a later step, usually metaphase I or metaphase II, to link completion of meiosis with fertilization. To identify regulators affecting developmental control of the meiotic cell cycle we screened for Drosophila mutants in which oocytes failed to undergo meiotic arrests or were unable to complete meiosis. We obtained mutants unable to release the prophase I arrest or the metaphase I arrest of the mature Drosophila oocyte. We also recovered mutants that exit these arrest points appropriately but fail to complete the meiotic divisions. We showed that one of these genes, cortex (cort), encodes a putative regulator of the Anaphase Promoting Complex/Cyclosome and acts specifically to control meiosis in the oocyte. The activity of CORT protein is regulated by translational control and degradation, and this may be a key aspect of developmental control of the meiotic cell cycle. At oocyte maturation, cort mRNA becomes polyadenylated and the protein first appears. Lengthening of the poly (A) tail is correlated with translation of a number of mRNAs in oogenesis and early embryogenesis. Following the completion of meiosis, CORT is targeted for degradation by the APC/C, and its mRNA becomes deadenylated, a likely mechanism to block further translation of the protein.

Regulation of early embryonic divisions by translational control: CORT activation of the APC/C leads to completion of meiosis and the degradation of the mitotic Cyclins A and B. These must then be translated to permit embryonic divisions. We identified a new protein kinase complex that is crucial for translation of Cyclin A and B protein at the onset of development. This kinase complex is composed of a ser/thr kinase catalytic subunit, PNG, and two activating subunits, PLU and GNU. The PNG kinase complex controls the oscillations between DNA replication and mitosis that occur in the rapid embryonic divisions by ensuring adequate levels of Cyclin B are translated. Sufficient levels of CyclinB lead to active CDK1/Cyclin B kinase (or MPF) that acts to block re-initiation and DNA replication and to initiate mitosis. PNG controls Cyclin B translation by poly (A)-dependent and independent mechanisms. Following the completion of meiosis, lengthening of the poly (A) tail on cyclin B mRNA is dependent on PNG, and this augments translation. During the embryonic cycles, Cyclin B translation is inhibited by the PUMILIO repressor. PNG acts antagonistically to PUM to permit Cyclin B translation, and these effects are independent of poly (A) tail length. Later in embryogenesis, PNG is crucial for the transition from maternal to zygotic control of development by promoting the translation of the SMAUG protein. SMG recruits the deadenylase complex to maternal mRNAs, leading to their degradation.

Differential replication as a developmental strategy: In addition to producing polyploid or polytene cells, Drosophila uses differential replication to optimize gene expression and metabolic efficiency. Heterochromatic regions are not replicated in polytene cells. Using microarrays to quantify genomic copy number we found that specific regions in the euchromatin are blocked from replication in polytene cells, and we are investigating the mechanism that prevents replication as well as the biological implication.
In the ovarian follicle cells two genomic regions containing eggshell protein genes are amplified, and the extra gene copies are essential for adequate gene expression. Using comparative genomic hybridization to microarrays, we identified four other amplified domains and showed that all contain genes expressed in the follicle cells. These amplicons are powerful model metazoan replicons, because they employ the normal replication machinery for multiple rounds of initiation, replication proteins can be visualized bound to the amplicons, and replication fork progression can be monitored directly. The developmental timing of replication initiation differs between the follicle cell amplicons, providing us the opportunity to define how differentiation signals can affect the recruitment of replication proteins and origin activation. These studies have uncovered two distinct mechanisms to regulate metazoan origin firing. One amplification origin is controlled by the Myb, Rb, and E2F transcription factors that interact directly with the Origin Recognition Complex. Another amplification origin, which fires later in follicle cell differentiation, requires transcription by RNA polymerase II to localize the essential MCM helicase and permit initiation.

Control of sister-chromatid cohesion and chromosome dynamics: Cohesion, or physical association, between the sister chromatids is essential for accurate kinetochore attachment to spindle microtubules and proper chromosome segregation. Cohesion is mediated in part by the cohesin protein complex. The cohesin complex is protected at the centromere and cohesion maintained by the MEI-S332 protein, the founding member of the Shugoshin protein family. We found that dissociation of MEI-S332 from the centromere at anaphase of mitosis and anaphase II of meiosis requires its phosphorylation by POLO kinase. MEI-S332 is controlled also by a multiprotein complex, the chromosome passenger complex. MEI-S332 physically binds the INCENP subunit, and phosphorylation of MEI-S332 by the Aurora B kinase subunit is needed for stable localization of MEI-S332 to the centromere.

The architectural changes necessary for proper chromosome segregation in meiosis require the sequential assembly of multi-protein complexes for sister-chromatid cohesion (the cohesin complex), for homolog synapsis (the synaptonemal complex), for chromosome condensation and sister-chromatid resolution (the condensin complex), and for spindle stability and cytokinesis (the chromosome passenger complex). We recovered a female-sterile mutation in the INCENP subunit of the chromosome passenger complex and female-sterile alleles of the dCAP-G subunit of the condensin complexes. These mutations reveal roles for these two complexes in the controlling the synaptonemal complex. INCENP is needed for maintenance of the synaptonemal complex in prophase I, whereas dCAP-G is required for its disassembly at the appropriate time. Both of these complexes play critical roles in chromosome orientation and spindle attachment during meiosis I.

Selected Publications

Vardy, L. and Orr-Weaver, T.L. The Drosophila PNG kinase complex regulates the translation of Cyclin B. Dev. Cell 12: 157-166 (2007).

Tadros, W., Goldman, A.L., Babak, T., Menzies, F., Vardy, L., Orr-Weaver, T., Hughes, T.R., Westwood, J.T., Smibert, C., and Lipshitz, H.D. SMAUG is the major regulator of maternal mRNA destabilization in Drosophila and is translationally activated by the PAN GU kinase. Dev. Cell 12: 143-155 (2007).

Resnick, T.D., Satinover, D.L. MacIsaac, F., Stukenberg, T., Earnshaw, W. C., Orr-Weaver, T.L., Carmena, M. INCENP and Aurora B promote meiotic sister chromatid cohesion through localization of the Shugoshin MEI-S332 in Drosophila. Dev. Cell 11: 57-68 (2006).

Ivanovska, I., Khandan, T., Ito, T., and Orr-Weaver, T. A histone code in meiosis: The histone kinase, NHK-1, is required for proper chromosome structure in Drosophila oocytes. Genes and Dev. 19: 2571-2582 (2005).

Lee, L. A., Lee, E., Anderson, M., A., Vardy, L., Tahinci, E., Ali, S.M., Kashevsky, H., Benasutti, M., Kirschner, M.W., and Orr-Weaver, T.L. Drosophila genome-scale biochemical screen for PAN GU kinase substrates identifies Mat89Bb as a novel, conserved regulator of the cell cycle and development. Dev. Cell 8: 435-442 (2005).

Clarke, A.S., Tang, T. T.-L., Ooi, D. L.-Y., and Orr-Weaver, T. POLO kinase regulates the Drosophila centromere cohesion protein MEI-S332. Dev. Cell 8: 53-64 (2005).

Claycomb, J., Benasutti, M., Bosco, G., Fenger, D. D., and Orr-Weaver, T. Gene amplification as a developmental strategy: Isolation of two developmental amplicons in Drosophila. Dev. Cell 6: 145-155 (2004).

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