Seeing is believing – Novel systems for studying chromosomal-derived circular DNA accumulation in human stem cells
Navn på bevillingshaver
Henrik Devitt Møller
Beløb
DKK 425,000
År
2017
Bevillingstype
Internationalisation Fellowships
Hvad?
The nuclear human genome consists of discrete linear chromosomes. However, extrachromosomal circular DNA elements (eccDNAs) can mutate out of the linear chromosomes and stay inside a cell for unknown periods of time. EccDNAs carrying proto-oncogenes are found to correlate with oncogenesis, likely through the accumulation of eccDNA copies within single cells. I will develop and exploit novel tools that will allow me to track specific eccDNAs inside single human stem-like cells. I intend to answer whether eccDNAs in general, accumulate in single cells and whether this has cellular consequences on e.g. cell morphology, cell-cycle duration, and cell viability. Furthermore, I will quantify the fate of eccDNA over multiple cell divisions to assess their stability inside living cells.
Hvorfor?
There is an unmet need for sensitive in situ techniques to monitor eccDNAs role inside cells, in order to learn more about their influence on the cell phenotype, their localization and prevalence over time. This is important as certain eccDNAs correlates with cancer progression, and eccDNA accumulation could be linked to ageing of cells. I will explore specific eccDNAs in human stem-like cells. Human stem cells are already used for medical treatments, and these cells are important for organ homeostasis as the cell steady-state depends on stem cell proliferation. Thus, potential preferential eccDNA accumulation in stem-like cell types may have undesirable, phenotypically consequences.
Hvordan?
The research will build upon the innovative research from Zürich (in vivo imaging in single cells), Copenhagen (eccDNA detection) and Aarhus (CRISPR technology), bringing leading research fields together for substantial synergistic effects that will facilitate state-of-the-art research on eccDNA inside living human stem-like cells. Tracking tools will include the well-established LacO/LacI sensor system, available in ETH Zurich, and development of a new, more sensitive tool for DNA imaging, using the RNA-Guided CRISPR-dCas9 system. Moreover, a biosensor will be developed that should offer further insight of eccDNA accumulation and allow easy quantitative measurement of eccDNA over time by fluorescence-activated cell sorting.