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 determine how an organism develops from a single-cell fertilized embryo to a multi-cellular adult. We want to define how developmental cues interface with DNA replication and chromosome segregation to link cell division to development. Using Drosophila as a model, we are investigating several developmental periods and types of cell cycles: meiosis and the production of gametes, fertilization and rapid early embryonic divisions, the transition to zygotic control of cell divisions during embryogenesis, and changes in gene copy number accompanying differentiation. Drosophila permits an interdisciplinary approach using genomics, genetics, cell biology, and biochemistry to discover new regulatory genes controlling DNA replication and chromosome segregation during development. Because these genes are conserved, Drosophila is an excellent model for identifying regulators that ensure proper human development and prevent cancer.
Gene amplification in Drosophila ovarian follicle cells visualized by EdU labeling. Each panel shows a single follicle cell nucleus, with the DNA labeled in blue and sites of nucleotide incorporation in red. On the left, six genomic regions of amplification are seen. In the mutant on the right, ectopic genomic regions undergo amplification. Images by Jared Nordman.
Differential DNA replication in development
Specific tissues in plants and animals, including humans, increase their genomic DNA content by becoming polyploid or polytene. Most Drosophila tissues become polytene as part of their differentiation program. In addition to these genome-wide increases in DNA content, we investigated whether differential DNA replication changes the copy number of specific genes. Increased copy number of genomic intervals is prevalent in cancer cells, yet the primary events causing gene amplification are unknown. Regions refractory to replication are thought to generate chromosome fragile sites that can lead to genome rearrangements in cancer.
We explored whether differential DNA replication leading to amplified or underreplicated genes occurs during development by performing Comparative Genome Hybridization (CGH) on tiled microarrays of genomic DNA isolated from differentiated polyploid or polytene Drosophila tissues versus embryos. We found genomic regions that are amplified as well as others that are underreplicated in specific tissues. These regions change copy number in specific types of differentiated cells; some are common between cell types whereas others show tissue specificity.
We are using these differentially replicated domains as models to define metazoan DNA replication origins, to elucidate how they are activated or repressed, and to determine how replication fork progression is affected by chromatin. This is proving a powerful model system to delineate the events that can lead to genome instability in cancer cells. In the ovarian follicle cells six genomic regions undergo amplification with precise developmental control of when and how many times each origin fires. We have shown that several distinct regulatory mechanisms are used to control initiation at the amplicons. Both origin firing and replication fork movement are affected by chromatin and genomic position. Our future efforts are directed towards defining the interactions between replication and chromatin proteins that influence origin activation and fork progression.
Polyploidization is a key mechanism to generate large cells in development. We recently found that the glial cells that make the blood-brain barrier in Drosophila increase their cell size by increasing DNA content, and this is required to maintain an intact envelope as the neuronal mass increases. Interestingly, the glia respond to neuronal mass to adjust their ploidy and resulting size. Thus an intact blood-brain barrier is present in brain tumor mutants because the glial cells increase their DNA content and size in response to the excess neurons.
Developmental control of meiosis and the onset of embryogenesis
Oogenesis accomplishes two developmental goals: halving the chromosome number by meiosis and differentiation of the oocyte. The differentiated oocyte is stockpiled with maternal components required to direct embryogenesis until expression of the zygotic genome. In most animals meiosis arrests first in prophase I to permit oocyte differentiation; a secondary arrest point coordinates completion of meiosis with fertilization. Release of both of these arrest points is accompanied by translation of stored maternal mRNAs, and we have shown that regulated proteolysis also is important for the completion of meiosis and restart of mitosis in the embryo. We used proteomic approaches to identify the proteins whose levels change at oocyte maturation (release of the prophase I arrest) and those that increase or decrease at egg activation (when release of the secondary arrest and completion of meiosis occurs). mRNAs recruited for translation at egg activation were defined by polysome profiling. In addition to identifying candidate regulators of the oocyte to embryo transition, these studies demonstrated that a meiosis-specific activator of the Anaphase Promoting Complex/Cyclosome, the product of the cortex gene, leads to targeted degradation of specific substrates at the completion of meiosis in the oocyte.
We identified the PAN GU (PNG) protein kinase complex that controls the onset of embryonic division and the transition from maternal to zygotic control by regulating translation. 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. Later in embryogenesis, PNG promotes the translation of the SMAUG protein, which recruits the deadenylase complex to maternal mRNAs, leading to their degradation.
Sher, N., Bell, G.W., Li, S., Nordman, J., Eng, T., Eaton, M.L., MacAlpine, D.M., and Orr-Weaver, T.L. Developmental control of gene copy number by repression of replication initiation and fork progression. Genome Res. 22: 64-75 (2012).
Unhavaithaya, Y. and Orr-Weaver, T.L. Polyploidization of glia in neural development links tissue growth to blood-brain barrier integrity. Genes and Dev. 26: 31-36 (2012).
Nordman, J., Li, S., Eng, T., MacAlpine, D., and Orr-Weaver, T. L. Developmental control of the DNA replication and transcription programs. Genome Res. 21: 175-181 (2011).
Kim, J. C., Nordman, J., Xie, F., Kashevsky, H., Eng, T., Li, S., MacAlpine, D., and Orr-Weaver, T. L. Integrative analysis of amplification in Drosophila follicle cells: Parameters of origin activation and repression. Genes and Dev. 25: 1384-1398 (2011).
Vardy, L., Pesin, J. and Orr-Weaver, T. Regulation of Cyclin A protein in meiosis and early embryogenesis. Proc. Natl. Acad. Sci. USA 106: 1838-1843 (2009).
Vardy, L. and Orr-Weaver, T.L. The Drosophila PNG kinase complex regulates the translation of Cyclin B. Dev. Cell 12: 157-166 (2007).
Pesin, J. and Orr-Weaver, T.L. Developmental role and regulation of cortex, a meiosis-specific Anaphase-Promoting Complex/Cyclosome activator. PloS Genetics 3: e202 (2007).