Cellular response to chromosomal aberrations
Elaborate mechanism are employed by each cell to faithfully propagate their genome and preserve a stable karyotype (constant number of chromosomes). In pathogenic situations however, loss or gains of chromosomes are often observed. These changes affect either a single chromosome (e.g. extra copy of chromosome 21 in Down syndrome) or a large number of chromosomes, which is frequently observed in cancer cells. Little is known about the impact of these chromosome gains or losses on cell physiology and how they promote the pathogenic state. In our laboratory we are using cutting-edge methodology to study the causes and consequences of karyotype changes.
Our Model System
To generate human cells carrying a single extra chromosome we use a technique called micronuclei mediated chromosome transfer (MMCT). To generate cells with extra copies of the whole sets of chromosomes (tetraploid cells) or with variable aneuploidy we treat the cells with drugs that interfere with cell division. Using state-of-the-art techniques including live cell imaging or flow cytometry we analyze how these cells propagate their abnormal genome. We also exploit genomics, transcriptomics and proteomics to determine the effects of the extra chromosomes on genome stability and protein homeostasis.
Impact of aneuploidy on genome stability
Using flow cytometry and live cell imaging we found that presence of extra chromosomes impairs proliferation of aneuploid cells. Global transcriptome and proteome analysis enabled identification of pathways that are deregulated in aneuploid cells irrespective of the identity of the extra chromosome. This analysis has revealed a strong down-regulation of DNA replication factors. Consistently, aneuploid cells show several hallmarks of replication stress. We are currently elucidating the mechanisms that contribute to replication stress in aneuploid cells.
Protein homeostasis in aneuploid cells
Cross-omic analysis by comparing genome, transciptome and proteome quantitative changes we found that genes from the extra chromosomes are normally transcribed and translated, but that expression levels of many proteins are adjusted to wild type levels. We study how autophagy, proteoasomal degradation or protein folding affect protein homeostasis in aneuploid cells and protein stability on a global scale.
Finding the Achilles heel of tetraploid cells
Sequencing of cancer genomes suggests that about 40 % of all cancers have undergone whole-genome doubling at some point during the tumorigenesis. In contrast, normally proliferating human cells rarely survive whole-genome doubling. Using RNAi and CRISPR/Cas9 we aim to determine factors that promote survival of tetraploid cell s in order to identify molecular targets that may be exploited for selective killing of cancers cells with abnormal karyotypes.
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Proteomics of DNA repair
DNA repair processes are indispensable for maintaining genome stability. During S-phase, DNA repair processes enable the replication machinery to bypass DNA lesions. Inactivation of these processes leads to developmental abnormalities and cancer (e.g. Fanconi Anemia). Combining mass spectrometry based proteomics with powerful in vitro DNA repair systems we determine DNA repair factors across DNA repair pathways and characterize their function at the molecular level.
Our Model System
To study DNA repair we use cell-free extracts derived from eggs of the African clawed frog Xenopus laevis. These extracts support efficient replication, condensation and segregation of chromosomal DNA, making them an ideal system to study chromatin-associated processes. They faithfully recapitulate several DNA repair pathways, yet allow unique as well as the repair of various DNA lesions.
To analyze DNA replication and DNA repair processes, we used the powerful in vitro DNA replication system based on Xenopus egg extract. The advantages of this versatile cell free system lie in the high degree of conservation of most essential cellular and molecular mechanisms, as well excellent synchronization of the DNA replication processes.
Using comprehensive proteomic profiling we have gained unprecedented insight into the temporal recruitment of known and novel DNA repair factors involved in bypassing psoralen DNA crosslinks. While some of these factors contribute to accurate repair of the lesions others play important roles in DNA damage signaling. Using established DNA repair assays we will pinpoint the exact role of these factors. Through expanding the palette of DNA lesions, our long-term goal is to gain a global view of DNA repair. Using additional proteomic techniques (mapping of phospho- or ubiquitylation sites) we aim to uncover the underlying regulatory mechanism of various repair pathways.