IFOM Chromosome Instabilities

Chromosome Instabilities

Research description

The ends of chromosomes are protected from unwanted enzymatic reactions by telomeres, structures composed of simple repeat sequences and associated proteins. In normal human somatic cells, telomere shortening during DNA replication eventually causes deprotection of telomeres, which activates DNA Damage Response (DDR) and halts cell division (replicative senescence). However, cells with abnormalities in the DDR pathway repeat the cell cycle, which leads to complete loss of their telomere sequences, and fusion between such chromosome ends. This fusion of chromosomes causes chromosome instability and is involved in carcinogenesis. On the other hand, the resulting cancer cells must somehow maintain their telomere length to avoid too much chromosome instabilities that cause cell death. Thus, the state of telomere protection is closely linked to cell survival and tumorigenesis. We have previously discovered a phenomenon in which cell cycle arrest in mitosis promotes telomere deprotection (mitotic telomere deprotection)(Figure 1) [MT Hayashi, et al., 2012, Nat Struct Mol Biol].

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Figure 1. Mitotic telomere deprotection.
IMR-90 human fibroblast cells were treated with microtubule drug vinblastine for 24 hours. Mitotic spread followed by staining of DNA damage marker gamma-H2AX (red), telomeres (green), and DNA (blue) revealed the deprotection of telomeres during mitotic arrest.

Furthermore, we found that, in cells that bypass replicative senescence, chromosome fusion resulting from telomere shortening induces spontaneous mitotic arrest, and that subsequent mitotic telomere deprotection induces cell death. These findings led us to propose a new pathway model that suppresses tumorigenesis [MT Hayashi, et al., 2015, Nature]. In recent years, however, a variety of phenotypes caused by chromosome fusion have been published, and it is not clear how the fate of chromosome fusion is determined in each cellular context. This is partly because previous studies failed to control the number and type of chromosome fusions (e.g., a fusion between two different chromosomes, a fusion between sister chromatids, and a fusion between short and long arms of the same chromosome). To circumvent this limitation, we have developed a cellular system called the Fusion Visualization system (FuVis) to visualize a single defined sister chromatid fusion in a living cell (Figure 2).

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Figure 2. A schematic for the Fusion Visualization system for Xp sister chromatid fusion (FuVis-XpSIS).
CRISPR/Cas9-targeting of the sister cassette induces spontaneous sister chromatid fusion due to erroneous repair, which results in full-length YFP expression through splicing. SA, splicing acceptor; SD, splicing donor; p2a, self-cleavage sequence; ex1 and ex2, exon1 and exon2 of YFP

The powerful FuVis system allows direct chasing of the fates of a single fusion at a single-cell level (Figure 3).

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Figure 3. Live cell imaging of the FuVis cells.
The FuVis cells express mCitrine (yellow triangle) upon induction of a defined Xp sister chromatid fusion. Live cell analysis revealed a micronuclei formation (white triangle) after the first mitosis following the mCitrine expression. Ph: phase contrast

The laboratory is now focusing on the molecular mechanisms of various phenotypes caused by chromosome fusions, including mitotic telomere deprotection as described above. We are especially interested in the effect of (1) different types of chromosome fusion and (2) cellular context, and (3) the mechanism of mitotic telomere deprotection as one of the phenotypes caused by chromosome fusions. We will expand the FuVis system to visualize different types of chromosome fusion in different cellular contexts. This unique approach will allow us to analyze the fates of chromosome fusion with unprecedented accuracy. Because chromosome fusion is at the core of chromosome instabilities and directly involved in tumorigenesis, potential outcomes will expand our comprehension of tumorigenesis from the unique viewpoint of direct interaction among cellular context, chromosome abnormalities, and tumor development.