Research Projects
Localization and cell cycle dynamics of the Drosophila ORC
Tina Baldinger
ORC is likely to function as the initiator protein in eukaryotes, i.e. its binding to chromosomal sites specifies the origins of bi-directional DNA replication. Drosophila melanogaster offers several distinct advantages for the analysis of replication initiation factors. Among them are the availability of a large number of hypomorphic variants of these proteins and an embryonic development which relies on maternally supplied stockpiles of replication factors. To analyze ORC’s binding to chromosomes in vivo, we generated transgenic flies expressing fully functional GFP fusions to Orc2, one of the ORC subunits. By genetic complementation of an Orc2-null background we could determine the subcellular localization of ORC throughout the cell cycle, in particular its chromatin association during mitosis (see figure). In combination with histone-RFP fusions this approach revealed changes in the dynamic behaviour of ORC in different tissues and throughout development. It turned out that the recruitment of ORC to its chromosomal sites is under the control of the major mediators of cell cycle cues, the cyclin dependent kinases. These observations will allow us to directly address the integration of the chromosome cycle in proliferation control, based on the use of a well established in vivo model.
Biochemical characterization of the human ORC
Anand Ranjan, Vishal Agrawal
We could co-express the genes for all six subunits of human ORC in insect cells and purify the resulting protein complex to homogeneity. Using a Xenopus in vitro replication assay, the functionality of this recombinant ORC was demonstrated. It turns out that human ORC is capable of forming various distinct sub-complexes, which differ in their stability and DNA binding properties. Interaction studies allowed us to determine critical features of the architecture of the human ORC. According to the Saccharomyces cerevisiae paradigm, ORC's binding to DNA is expected to be ATP dependent. We are currently investigating if this unusual mode of regulating sequence specific DNA interactions is connected to the ATP dependence of specific subunit interactions that we observed in our in vitro assays. To this end we are also testing the biochemical properties of recombinant human ORC defective in ATP interactions and extend these studies to human cell cultures. The goal of this project is a better understanding of the mechanisms by which homologous initiator proteins like those of yeast and humans accomplish highly divergent modes of origin determination.
Ablation of preRC proteins
Ibrahim Kocman
ORC binding to chromatin is the first step in the assembly of the large, origin-associated prereplicative complex, preRC. This process renders chromosomes replication competent. All preRC proteins are essential for DNA replication and thus also for cell proliferation. As such they constitute a potential target for anti-proliferative therapies, e.g. in the treatment of cancer. Based on our biochemical analyses we were able to identify among the preRC genes promising candidates, which should be particular well suited as a target. In collaboration with Silence Therapeutics, Berlin, we investigate the effects of siRNA mediated knockdown of the expression of such genes, which results in the inhibition of DNA synthesis as well as the stalling of cells in mitosis. We are currently evaluating the use of this technology for the control of cell proliferation in animal models.
Promoter crosstalk and epigenetic regulation of transgenes
Mathias Hampf
Transcription units randomly integrated in the chromosomes of mammalian cells are subject to both epigenetic control and the influence of nearby transcription signals. These findings have important implications for the design of gene expression vectors for transgenesis and gene therapeutic approaches. It is often desirable to transfer multiple transcription unit in one step. We are analyzing the effects these transgenes exert on each other by using an inducible transcription system. Upon induction of a target gene, a neighbouring "constitutive" transcription unit can be co-regulated depending on the nature of the promoters used. Vice versa, these promoters can have a dominant influence over the characteristics of the inducible transcription unit. To understand the mechanisms of this crosstalk we need to control epigenetic effects on transgene expression, like DNA methylation and chromatin compaction. We recently established protocols allowing for the reproducible generation of high level expressing stable cell lines, that can escape epigenetic downregulation over prolonged periods of time. These cells also show uniform expression when analyzed on the single cell level. In combination with site-specific recombination protocols and chromosomal engineering this approach should allow us to design predictable transgene expression protocols inert to epigenitic distortions and might also permit gaining novel insights into the principles governing the expression of endogenous genes.