Principal Investigator Terry Orr-Weaver
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. 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, and these changes in gene copy number correlate with whether the genes are highly expressed or not expressed, respectively.
We are using these differentially replicated domains as models to define metazoan DNA replication origins and to elucidate how they are activated or repressed. 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 identified two new metazoan DNA replication origins from these amplicons. Several distinct regulatory mechanisms are used to control initiation at the amplicons. One amplicon contains a replication enhancer that activates an adjacent origin, in part by recruiting the Origin Recognition Complex (ORC). Initiation is controlled by the Rb/E2F and Myb transcription factors via direct interaction with ORC at the replication enhancer. The origin of a second amplicon is activated by transcription, which is necessary for loading the essential MCM replicative helicase. Initiation at a third amplicon is independent both of transcription and ORC. This diversity of control mechanisms suggests that in mammalian cells multiple regulatory mechanisms also exist and that their failure may lead to gene amplification in cancer cells.
We also are exploiting the follicle cell amplicons to decipher the control of replication fork movement in metazoans. We identified mutants with increased rates of fork elongation at these amplicons. One of these is a missense mutation in cyclin E, uncovering a previously unrecognized regulatory role at the replication fork. These mutants additionally lead to ectopic sites of gene amplification, identifying genomic regions vulnerable to repeated rounds of replication initiation.