DNA replication initiates at specific sites known as origins. However, the mechanisms multicellular eukaryotes use to define origins are a mystery. Evidence suggests that chromatin structure may play an important role in origin specification. The 3rd chorion amplification origin in Drosophila is a model for understanding the relationship between DNA replication and chromatin. The 3rd chorion origin is one of the best defined origins in metazoans, and several powerful cell biological and molecular assays exist to quantifiy origin function. Published data from our lab shows that nucleosomes at this origin are hyperacetylated, which is important for origin function. The broad goals of this proposal are to provide insight into the fundamental principles governing the relationship between DNA replication and chromatin structure, two processes which are both implicated in cancer progression. In the long term, these fundamental principles will be applied to our understanding of the basic cellular processes that, when disrupted, can result in cancer.
Aim I. Identification of the acetyltransferase required for amplification. The appropriate acetyltransferase will be identified using a variety of approaches. Reducing active levels of acetyltransferase will verify a role in 3rd chorion origin function.
Aim 2. Identification of the cis sequences required for 3rd chorion acetylation. In this aim, the relative requirements of cis sequences and chromatin modification on 3rd chorion origin specification will be examined. Specific deletions within the origin will be tested for an effect on acetylation and origin function.
Aim 3. Determining the effect of nucleosome acetylation on ORC localization. The Origin Recognition Complex (ORC) binds directly to origins and is critical for origin function. Flies with reduced acetyltransferase activity will be tested for an effect on ORC localization to the 3rt chorion origin. Relevance: Misregulation of DNA replication can lead to genomic instability, a common characteristic of cancer cells. The maintenance of proper chromatin structure is also implicated in cancer. By using a well-defined origin in fruit flies as a model system, fundamental principles regarding the relationship between chromatin and replication will be discovered. Additionally, factors associated with this model origin have been directly implicated in the human cancers myeloblastosis and retinoblastoma. By better understanding the basic processes implicated in cancer, better diagnostics and treatments can be developed.
|McConnell, Kristopher H; Dixon, Michael; Calvi, Brian R (2012) The histone acetyltransferases CBP and Chameau integrate developmental and DNA replication programs in Drosophila ovarian follicle cells. Development 139:3880-90|
|Liu, Jun; McConnell, Kristopher; Dixon, Michael et al. (2012) Analysis of model replication origins in Drosophila reveals new aspects of the chromatin landscape and its relationship to origin activity and the prereplicative complex. Mol Biol Cell 23:200-12|