FOR2800

Cancer is a disease of the genome, with both structural and numerical chromosomal aberrations, so called aneuploidy, occurring concomitantly in most tumors. Despite this co-occurrence, the advances in the characterization of point mutations and structural and numerical changes failed to provide an insight into a possible functional relationship between structural and whole chromosomal instability. Recently, novel evidence suggests that numerical chromosomal aberrations leads to delayed S phase ...

We intend to shed light on the interplay between the regulation of replication origin firing and mitotic chromosome segregation. The relationship between faithful genome replication and chromosome segregation is mutual: de-regulated genome replication causes replication stress and chromosome missegregation [1, 2]. Conversely, chromosome missegregation causes replication stress [3, 4]. Although it is clear that proper regulation of origin firing, the main focus of our lab, is key for complete and ...

To maintain genome stability, human cells depend on accurate DNA replication in S-phase and chromosome segregation in mitosis. Replication stress refers to slowing down or stalling of replication forks that occurs due to DNA lesions, R-loops or oncogene activation. One consequence of replication stress is entry of cells into mitosis with incompletely replicated DNA and thus aberrant mitosis. Phosphorylation of proteins by kinase ATR and its downstream effector CHK1 protects stalled replication ...

Replication stress is a key driver of genomic instability giving rise to human disease, for instance cancer or developmental disorders. It is caused by a variety of defects such as lesions blocking replication (e.g. DNA interstrand crosslinks) or processes that lead to the exhaustion of DNA precursors. This generates under-replicated DNA and replication intermediates that can be rescued by repair processes requiring homologous recombination between sister chromatids. Often, cells exposed to ...

The objective of the central project is providing access to state-of-the-art sequencing technology and methods as well as novel next-generation sequencing (NGS)-based approaches to all partners and individual projects within the consortium. Advances in NGS and NGS-based approaches such as whole-genome sequencing (WGS) have tremendously improved the detection of structural variants/copy number variations (CNVs) and single somatic variants and mutations as measures of genome integrity. With ...

Replication stress is one of the major causes for chromosomal instability which is a hallmark of cancer. Incomplete repair can lead to severe mitotic defects and ultra-fine anaphase bridges (UFBs) are a typical consequence. So far, five different classes of UFBs have been discribed: fragile sites-UFBs, centromeric-UFBs, telomeric-UFBs, homologous recombination-UFBs and rDNA-UFBs, which can be distinguished by their location and the way how they arise. Although previous studies have already proposed ...

Genomic instability is a hallmark of cancer and can be frequently induced by replication stress. Incomplete repair of replication intermediates can lead to persistent linkages between sister chromatides, detectable during mitosis as ultra-fine anaphase bridges (UFBs). The active resolution of UFBs via several UFB factors are crucial to prevent mitotic catastrophe, chromosomal missegregations or aneuploidy. Previous studies have already revealed that UFBs are processed by a distinct set of proteins, ...

Many cancer cells are chromosomally unstable, a phenotype describing a tendency for accumulating chromosomal aberrations. Entire chromosomes tend to be gained or lost, which is called whole chromosome instability (W-CIN). Structural chromosomal instability (S-CIN) describes an increased rate of gaining, losing or translocating smaller parts of chromosomes. Here we analyse data from 33 cancer types to find differences and commonalities between W-CIN and S-CIN. We find, that W-CIN is strongly linked ...

Cancer researchers perform experiments with cell lines to better understand the biology of cancer and to develop new anti-cancer treatments. A prerequisite to translate promising results from these \textit{in vitro} experiments to clinical applications is to use the most appropriate cell line for a given tumour or cancer subtype. We present MFmap (model fidelity map), a deep learning technique to integrate cancer genomic data from patients with cell line data. The MFmap neural network compresses ...

Chromosomal instability (CIN) is a hallmark of cancer and comprises structural (S-CIN) and numerical CIN (W-CIN). Recent work indicated that replication stress (RS), known to contribute to S-CIN, also affects mitotic chromosome segregation, thereby possibly explaining the common co-existence of S-CIN and W-CIN in human cancer. Here, we show that RS-induced dormant origin firing is sufficient to trigger W-CIN in human cancer cells. We discovered that overexpression of origin firing genes including ...

One of the main characteristics of BLM syndrome patients and BLM deficient cell lines is the enhanced occurence of sister chromatid exhange, detectable in metaphase chromosome spreads. Successful BLM depletion in our homozygous BLM-AID cell line was accompanied by a significant increase of sister chromatid exchange (SCE).

To study how mitotic functions of BLM helicase contribute to genome stability, we genereated chromosomally stable HCT116 cell lines, in which BLM protein can be rapidly degraded in a cell-cycle specific manner. Using the CRISPR/Cas9 technology, a homology donor encoding an auxin-inducible degron (AID), GFP and a selection marker was inserted at the last coding exon of the endogenous BLM gene. To generate the homology donor, a modular cloning system was set up using the Golden Gate assembly strategy. ...

FANCD2 ChIP-MS experiments in refined synchronized HCT116 cells with different types of replication stress are performed to identify additional proteins involved in the resolution of UFBs. Through reciprocal ChIP-MS and colocalization analysis by immunofluorescence experiments observed interactions will be validated. In collaboration with SP-Z promising candidates will be prioritized and subsequently tested for their contribution to the resolution of UFBs and chromosome segregation. In collaboration ...

In this study we can investigate the ability to pull down BLM in BLM-AID cells with its inserted GFP tag and use the BLM-depleted cell line as a control. The ChIP-MS analysis of BLM in synchronized cells can reveal new interaction partners invovled in the resolution of UFBs. On the other hand pulling down other UFB binding proteins in BLM depleted cells can help to understand the impact of cell-cycle specific BLM depletion on UFB resolution.

We implemented the pan-cancer chromosomal instability association studies pipeline using R-3.6. The source code and data are available at https://github.com/mcmzxx/pancan_cin

CHIP-MS was performed with HeLa cells carrying a bacterial artifical chromosome for the expression of GFP-tagged PICH under it's endogenous promotor. Cells were treated with drugs to induce different types of ultra-fine bridges (UFBs). Treatments included: a) 24h exposure to 0.1 uM Aphidicolin, b) 24 hours exposure to 120 ng/ml mitomycin C, c) 6.5 hours incubation in 0.05 ug/ml nocodazol followed by a release into 0.1 uM ICRF-193 topoisomerase inhibitor.