SP-6: Proteomics of mitotic inter sister chromatid junctions

Our Research Unit addresses an important question on the origin of chromosome instability (CIN), which causes structural as well as numerical chromosome aberrations. Importantly, CIN and increased levels of chromosome aberrations are closely associated with many human diseases including cancer, neurodegenerative diseases and age-related syndromes and can act as key drivers for disease development and progression. An important condition that causes structural chromosome instability (S-CIN) leading ...

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, ...

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.

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.